Training Manual For Food Testing, Microbiology and Physical Chemistry Course UPDATED 33

Dr.
Ali Mohamed Ali Iye ( Ali kanu)

Food Safety Consultants P. O. Box 7698-00100 Nairobi KENYA Tel, 0722809835, 0710398280 E. mail: fsc.consultants2000@gmail.com
TRAINING MANUAL OF COURSE ON FOOD MICROBIOLOGY AND PHYSICAL CHEMISTRY TECHNIQUES HELD ON JANUARY 2013
VENUE: IGAAD SHEIKH TECHNICAL VETERINARY SCHOOL
Prepared and conducted by: Mr. Joseph Kimari and Mr. Duncan Ndegwa of food safety consultants
1 Mr. Duncan Ndegwa Mr. Joseph Kimari and
Dr.Ali Mohamed Ali Iye ( Ali kanu)
Table of Contents
1.0 GOOD LABORATORY PRACTICE.........................................................................4 2.0 LABORATORY SAFETY......................................................................................4 3.0 PERSONAL HYGIENE........................................................................................4 4.0 LABORATORY DOS AND DONTS..................................................................5 5.0 BACTERIOLOGY..............................................................................................6
5.1 GENERAL IDENTIFICATION OF BACTERIA.........................................................6 5.2 IDENTIFICATION OF BACTERIA....................................................................7 5.2.1 Gram Staining .............................................................................7 5.3 MEDIA USED IN BACTERIOLOGY...................................................................8 5.4 MEDIA PREPARATION PROCEDURE ...............................................................9 5.5 SAMPLE PREPARATION AND DILUTIONS.........................................................10 5.5.1 Procedure for sample preparation and dilution.................................11 5.6 TOTAL VIABLE COUNTS (TVC)..................................................................13 SEE PROCEDURE BELOW...............................................................................14 PROCEDURE FOR PERFORMING TOTAL VIABLE COUNTS IN MILK, DAIRY PRODUCTS, FOODS AND SWABS BY THE POUR PLATE METHOD.........................................................14 5.7 COLIFORM COUNTS ( PLATE METHOD )......................................................17 PROCEDURE FOR THE ENUMERATION OF COLIFORM ORGANISMS BY THE POUR PLATE METHOD.................................................................................................18 5.8 YEAST AND MOULDS COUNTS...................................................................22 5. 9 MOST PROBABLE NUMBER TESTING ( MPN) IN WATER....................................25 5-10. DETECTION OF SALMONELLA SP..............................................................28 PROCEDURE FOR THE DETECTION OF SALMONELLA SP. IN ALL FOODS, MILK, AND DAIRY PRODUCTS & ENVIRONMENTAL SWABS..............................................................29 ..........................................................................................................29 5.11 ENUMERATION OF STAPHYLOCOCCUS AUREUS...............................................33 PROCEDURE FOR THE ENUMERATION OF STAPHYLOCOCCUS AUREUS............................34 5.12 ACIDITY TEST - TITRATABLE ACIDITY OF MILK.............................................38 5.13 BUTTERFAT FAT CONTENT GERBER METHOD ...............................................40 5.14 RESAZURIN TEST................................................................................42 5.15 ALCOHOL TEST .................................................................................45 5.16 MOISTURE ANALYSIS IN FOODS...............................................................46
Procedure for the Determination of Moisture content by Ohaus MB45 Thermo gravimetric Method .............................................................................................................47
5.17 PHOSPHATASE TEST FOR PASTEURIZED MILK...............................................48 2 Mr. Duncan Ndegwa Mr. Joseph Kimari and
Dr.Ali Mohamed Ali Iye ( Ali kanu) 5.18 ANTIBIOTIC RESIDUE IN MILK AND MEAT/LIVER...........................................50 5.18.CHLORINE CONTENT IN WATER...............................................................53 5.19 0BRIX.............................................................................................54 5.20 PROCEDURE FOR DETERMINATION OF HYDROGEN PEROXIDE IN MILK...................55 ANNEX 2 MAKING STERILE BLOOD AGAR PLATES.................................................60 ANNEX 3 MAKING MCCONKEY AGAR...............................................................62 ANNEX 4 PERFORMING THE CATALASE TEST......................................................63 ANNEX 5 DETECTING INDOLE PRODUCTION.......................................................64 ANNEX 6 IDENTIFYING OXIDASE POSITIVE ORGANISMS.........................................65 ANNEX 7 MAKING STERILE UREA AGAR............................................................66 ANNEX 9 PROCEDURE FOR MAKING TRIPPLE SUGAR IRON......................................69 ANNEX 10 PROCEDURE FOR MAKING STERILEBAIRD PARKER MEDIUM....................71
1.0 GOOD LABORATORY PRACTICE

Good Laboratory Practices are generally accepted methods to perform activities or operations in the laboratory. These practices are known or believed to be safe and protects the workers and have a positive influence on the quality of the result. Good Laboratory Practice (GLP) embodies a set of principles that provides a framework within which laboratory work is planned, performed, monitored, recorded, reported and archived. The approach is meant to cover good laboratory practice as far as safety, personal hygiene and good working practices are concerned.
2.0 LABORATORY SAFETY

The most important factor in the prevention of laboratory acquired infections/accidents/incidences and maintenance of laboratory standards is good laboratory practice. Safety in the laboratory is of paramount importance and all employees have a duty to take reasonable care for the health and safety of themselves and all other persons who may be affected by their acts or omissions at work. The most efficient means of achieving this is to spend some time identifying all safety hazards in a particular laboratory, assess them and the means of avoidance or control determined. This together with good laboratory practice (GLP) will ensure a safe working environment and improve standards of activities in the laboratory.
3.0 PERSONAL HYGIENE

Apart from good laboratory practice individual workers can contribute to the prevention of self-infection and the infection of others by personnel hygiene. To prevent infection in the laboratory, personnel have to identify the routes of infection, most hazardous organisms that requires use of biosafety cabinet, different routes of infections, which techniques are dangerous and how a worker can protect himself. Below, is a list of laboratory dos and donts;
4.0 LABORATORY DOS AND DONTS

1. Always wash your hands before starting work, after working and always wear a laboratory coat while working in the laboratory. 2. Always wear gloves for procedures necessitating direct contact with infectious materials. 3. Mouth pipetting is prohibited. 4. Always swab the workbench with 70% alcohol or any other suitable disinfectant after each working session. 5. Always disinfect and wash your hands after handling infectious materials, before eating or drinking during breaks in the day, when using the telephone and when you leave the laboratory. 6. All spills, accidents and potential exposures to infectious materials must be reported to a senior member of staff and entered in the ACCIDENT BOOK. Always keep the laboratory neat, clean and free from materials not related to your work. 7. Eating, drinking, smoking, storing food, chewing pencils, biting nails or applying cosmetics are not permitted in the laboratory working area. 8. All visitors must be signed in and wear laboratory coats when they enter the laboratories. 9. Persons who are at increased risk of acquiring infection, e.g. children allowed to the laboratory. are not

10. Cuts, scratches, sores and other lesions on the hands and exposed parts of the body should be covered with adhesive plasters.
5.0 BACTERIOLOGY
5.1 General identification of bacteria
The most common bacteria in human, veterinary and food microbiology can be classified into three distinctive shapes; a) Cocci ( spherical )
b) Bacilli ( rod shaped )
c) Vibrio ( comma shaped ) The cocci can be further divided into; a) Diplococci b) Streptococci ( chains ) c) Staphylococci ( clusters )
5.2 Identification of bacteria

The first step in the identification of bacteria is the performance of the Gram stain.
5.2.1 Gram Staining

The purpose of this procedure is to differentiate bacteria into Gram negative and Gram positive organisms. The Grams reaction is due to differences in their cell wall structure. The reporting should include the following information: The Gram reaction of the bacteria, whether Gram positive or Gram negative. Morphology of the bacteria, whether cocci, diplococci, rods, coccobacilli or yeast cells. Note:Cocci are round or oval bacteria measuring about 0.5 to 1 m in diameter. Rods are stick like bacteria with rounded tapered, square or swollen ends. They measure 1 to 10m in length by 0.3 to 1 m in width. The short rods with rounded ends are often called coccobacillus. Rods and cocci are sometimes found in chains, and this should be mentioned when describing the bacterial morphology. Discussion to include the principle of the test, interpretations and presentation of results. Procedure: 3.1 Make a smear on the slide by emulsifying a sample of the colony in normal saline, for purulent specimens spread the material thinly using a wire loop and for a swab roll the swab on the slide. Allow the smear to air dry. Heat fix. 3.4 Cover with crystal violet for one minute and wash in tap water. 3.5 Cover with Lugols iodine for one minute and wash. 3.6 Decolourize rapidly with acetone and wash immediately. 3.7 Counter stain with Safranin for two minutes and wash. 3.8 Wipe the back of the slide, and place in a draining rack to air dry.
3.2 3.3

3.9 Examine using oil immersion.
Observations: Gram positive bacteria..... Dark purple Yeast cells....... Dark purple Gram negative bacteria......Pale to Dark red After establishing the gram reaction of an organism further characterization is done using biochemical tests. The biochemical tests mostly applied in a Food Microbiology laboratory are as follows; Catalase Coagulase Indole production Oxidase Dnase Testing for urea Tripple sugar iron API profiles etc
5.3 Media used in bacteriology

The main types of culture media are: a) Basic e.g. Nutrient agar b) Enriched or enrichment eg Blood agar c) Selective e.g. XLD d) Differential e.g.MaConkey agar e) Transport e.g. Stuarts Transport media The different types of media and their application will be discussed in detail. A wide variety of media are available for cultivation of pathogenic bacteria and fungi. Most are available commercially in the dehydrated form. The formulations and directions for the preparation of the commercially available dehydrated media are normally given as per manufacturers instructions
5.4 Media preparation procedure

The purpose of this procedure is to ensure that media production is standardized, organized and recorded to ensure that media components are available when required and that the media used in analyses is suitable for use, traceable back to production, components, methods used and that production operators are identifiable if necessary. Production of sterile media is important for proper testing of various types of samples. Failure to produce media as per the laid down work instructions would lead to production of media which may not sustain the growth of the target organisms. Great care is therefore necessary in this area. The water used in the preparation of culture media should be distilled or de-ionized. The water should have a conductivity of < 15 micro-Siemens and a pH of not less than 5.5 and not more than 7.7. If the de-ionization process is inadequate, residue acid may cause the final pH of the medium to drop. Media should be tempered in water bath @ 45 oC. The temperature of the water bath should be continuously monitored with a thermometer. Note: A. If pathogens are to be isolated successfully, culture media must be prepared carefully. Each of the following steps must be performed correctly; o Weighing and dissolving o Addition of heat sensitive ingredients o Dispensing o Sterilization and sterility testing o pH testing o Quality control o Storage B: Record the day of opening a new bottle of media

Dehydrated media is hygroscopic, i.e., it absorbs water. Dehydrated media should be weighed rapidly and tops replaced immediately and tightly Use completely clean glassware Use distilled water or deionized water If possible check the Electrical conductivity of the distilled water When heating is required to dissolve the medium, stir while heating and control the heat to prevent boiling and foaming which may damage the medium. Overheating a medium can alter its nutritional and gelling properties, and its pH. Practicals Preparation of severalbatches of standard plate count agar; Buffered Peptone Water, Violet Red Bile Agar, MacConkey Agar, XLD and BGA weighing, autoclaving, tempering, pouring and storage. Several batches of media were prepared and training on the all above aspects applied. In addition the following was covered; a) Autoclaving b) Media tempering c) Media pouring d) Quality control; to assess the performance of the media The following procedures in microbiology and physical chemistry testing in foods were discussed and practicals done;
5.5 Sample preparation and dilutions

The procedure describes how to prepare a 10 -1 homogenate of food samples in a suitable diluent for enumeration purposes and preparation of further dilutions for enumeration in samples likely to contain high numbers of organisms. For homogenous samples including powders and free flowing liquids and concentrate mix well before removing a portion for testing. Do not shake powders immediately before testing as the environment may become contaminated by dust particles. For heterogeneous samples such as sandwiches it is usually appropriate to remove a representative portion of the whole product so that all components are taken.

Using sterile instruments and aseptic technique, weigh a representative 25g sample of each food into either a sterile bottle. Aseptically add 225 ml of sterile buffered water or the appropriate diluent according to the method. See procedure below.
5.5.1 Procedure for sample preparation and dilution

1. Purpose The procedure describes how to prepare a 10 -1 homogenate of sample in a suitable diluent for enumeration purposes and preparation of further dilutions for enumeration in samples likely to contain high numbers of organisms 2. Scope This procedure is applicable to the microbiological examination of food samples 3. Responsibility The technician in-charge of the Bacteriology laboratory is responsible for the implementation of the procedure. Requirements: Weighing balance 0.01 Sterile spoons Mechanical blender Bunsen burner Vortex Mixer Appropriate diluents 1000 l automatic pipette Sterile 1 ml pipette tips Reference Documents: Preparation of samples and dilutions 03/05/2005 Reference no: F 2 Health Protection Agency, Standard Units, UK
4.
5.
6. Procedure 6.1 Sample preparation For homogenous samples including powders and free flowing liquids and concentrate mix well before removing a portion for testing. Do not shake powders immediately before testing as the environment

may become contaminated by dust particles. For heterogeneous samples such as sandwiches/samosas it is usually appropriate to remove a representative portion of the whole product so that all components are taken. 6.2 Preparation of homogenate Using sterile instruments and aseptic technique, weigh a representative 25g sample of each food into either a sterile bottle. Record the weight. For samples such as meat aseptically blend samples using a Mechanical blender. Blend at 10,000 12, 000 rpm for 2 minutes.
6.4
Add exactly nine times the weight or volume of Buffered Peptone Water at ambient temperature to give a 1 in 10 (10 -1) suspension. Record the weight or volume used. If the amount of food available is less than 25g maintain the sample: diluent volume ratio at 1:9 (1 in 10dilution). Using a mechanical blender to homogenize the suspension (See 6.2 above) The time lapse between preparation of the homogenate and inoculation of the counting media should not exceed 45 minutes. Buffered peptone water (BPW) is used for the preparation of the homogenate when a single 10-1 homogenate is made for both detection of Salmonella and enumeration of other organisms. In this instance prepare the homogenate by weighing at least 27g of sample, add an appropriate volume of BPW, remove 20mL for enumeration and use the remainder of the homogenate for detection of Salmonella. Preparation of dilutions. Use Buffered peptone water (BPW) at ambient temperature for all dilutions.
6.5
6.6
6.7
6.8

6.9 To prepare decimal dilutions transfer 1.0ml of the 10 -1 homogenate to 9.0mL of BPW avoiding contact between the pipette/pipette tips, the diluent and the inside wall of the container. Mix carefully using a vortex mixer for 5-10 seconds. This constitutes the 10 -2 dilution. Using a fresh pipette/pipette tip for each dilution repeat this procedure to produce further decimal dilutions.
6.11
5.6 Total viable counts (TVC)

This procedure ensures that the number of colony forming units (cfu) per millilitre or per gram of an original sample is determined. A defined test portion or series of decimal dilutions of the sample are mixed with culture media in Petri dishes and incubated. The number of cfu per millilitre or per gram of the original sample is calculated from the number of colonies counted on selected dishes. The calculation is carried out using a formula described in the procedure for TVC. The test is carried out on raw milk, milk products, food (eg meat) and animal feeds. The TVC gives you the levels of contamination of the food and hence the quality of the food. The media for performing this test must be cooled down to 45oC. Higher temperatures of the media will kill the bacteria. TVCs of products are useful for indicating the overall microbiological quality of products and potential spoilage in perishable products. National bodies have specifications on various products and to establish whether a product passes TVC have to be carried out. In products where the bacterial load is expected to be high, decimal serialdilutions needs to done to determine the breakpoint. Plate with counts between 10 and 300 will be used for calculation. Plates with >300 colonies will considered as uncountable. After counting the colonies on the Petri dishes, the calculation of the final count is as follows; N= Where C/ [V (n1 + 0.1n2) d] = n1 = sum of all colonies on all Petri dishes counted number of dishes in the first dilution counted

n2 = number of dishes in the second dilution counted
d =dilution for which the first counts were obtained V= volume applied to each dish
Example Volume applied 1 ml -2 Dilution 1/100 (10 ) 278 and 290 colonies -3 Dilution 1/1000 (10 ) 33 and 28 colonies N=(278+ 290 + 33 + 28)/1 x (2+[0.1 x 2]) x0.01] = 629/0.022 = 28590 or 2.9 x 104 cfu/ml See procedure below. Procedure for Performing Total Viable Counts in milk, dairy products, foods and swabs by the Pour Plate Method. 1.Purpose This procedure ensures that the number of colony forming units (cfu) per milliliter or per gram of an original sample is determined. A defined test portion or series of decimal dilutions of the sample are mixed with culture media in Petri dishes and incubated. The number of cfu per milliliter or per gram of the original sample is calculated from the number of colonies counted on selected dishes. 2 .Scope This covers the use of the test for milk, dairy products and swabs in the Bacteriology Laboratories. 3. Responsibility The Laboratory Head is responsible for the correct implementation of the procedure

4. Requirements Weighing balance (0.01g) Spatula Autoclave Autoclave tape Bunsen burner Vortex Mixer Incubator @ 30oC 1oC Water bath @ 45oC 1oC Conical flask (250 mls, 500mls or 1000mls) 1000 l automatic pipette Sterile 1ml pipette tips 9ml sterile quarter strength Ringers solution or Buffered Peptone Water in o Universal bottle. Autoclaved @ 121 C for 15 minutes. This is used as the diluent. Sterile Petri dishes Sterile Standard Plate Count Agar in a sterile conical flask (CM0 463). 5. Reference Documents: Plate count test at 30oC 03/05/2005 Reference no: D2 Health Protection Agency, Standard Units, UK 6. Procedure: 6.1 Prepare the sample as described in the Procedure for preparation of samples and dilution. The interval between mixing and pipetting should not exceed 3 minutes. 6.2 Transfer 1ml of the sample aseptically into 9ml of sterile diluent in a Universal bottle and mix thoroughly. This is the Primary Dilution. 6.3 Transfer 1ml from the Primary dilution (6.2 above) aseptically using a fresh sterile pipette tip to a further 9ml of diluent and mix thoroughly. Further dilutions are prepared by transferring 1ml of each successive dilution into a further 9 ml. of diluent using a fresh sterile pipette tip in each case. 6.4 Transfer 1ml of each chosen dilution using a sterile pipette tip into labeled sterile Petri dishes starting with the most dilute of the dilutions prepared. 6.5 Add 15 18 ml of the tempered melted medium aseptically to each inoculated Petri dish.

6.6 Mix the contents of the Petri dish immediately after pouring by 5 to and fro movements of the dish followed by 5 circular clockwise movements followed by 5 to and fro movements at right angles to the first set, followed by 5 circular anti-clockwise movements. Allow the Petri dishes to stand on a clean horizontal surface until the medium sets, invert and transfer to the incubator. Incubate the Petri dishes at 30oC 1oC for 72 3 hours
6.7 6.8
For each batch of agar, pour one Control Plate per every1/2 litre of medium (a sterile Petri dish) and incubate with the sample plates. If there is bacterial growth and colonies are observed the test results should be considered with caution and entered into the Day Book .If more than 10 colonies are observed the test results are void. The demonstration of growth in the control plate(s) must be reported to the Head of Bacteriology and action taken that he recommends.
7.0
Expression of Results Calculate the number of cfu, N, per millilitre of sample as follows:N= C/ V [(n1 + 0.1n2) d] Where =sum of all colonies on all Petri dishes counted. n1 = number of dishes in the first dilution counted n2 = number of dishes in the second dilution counted d = V = dilution for which the first counts were obtained volume applied to each dish
Example Volume applied Dilution 1/100 (10-2) Dilution 1/1000 (10-3)
1 ml 278 and 290 colonies 33 and 28 colonies

N = (278+ 290 + 33 + 28)/[1 x (2+ {0.1 x 2}) x0.01] = 629/0.022 = 28590 or 2.9 x 104 cfu/ml Enter the results in the Laboratory Day Book showing the calculation Reporting of result Record the count expressed as two significant figures and expressed as a power of 10. When the third figure is less than five, do not change the preceding figure, when the third figure is 5 or more increase the preceding figure by one unit. e.g 28,500 is expressed as 2.9 x 104 If the plate prepared from the 10-1 .dilution contain no colonies, report the number of organisms as less than 1.0 x 10 1per ml or g (derived from 1 x 1/d, where d the dilution is 10-1 ). If there are only plates containing more than 300 colonies report the count as greater than 3.0 x 10 2 per ml or per g multiplied by the dilution factor. For example, if -3=u/c -4=u/c -5=u/c , the count will be > 300x10 5 = 3x108 CFU/ml. If all plates have uncountable colonies report as being an Estimated count based on the highest dilution measured. If a sample is plated undiluted, and no growth appears on plate, report as Nil CFU/ml .
8.0
Records Bacteriology Laboratory Day Book.
5.7 Coliform counts ( plate method )

This procedure ensures the identification and enumeration of coliform organisms (E. coli, Citrobacter, Enterobacter or Klebsiellaspp.). Coliforms are indicators of external contamination. Coliform organisms are able to ferment lactose within 48 hrs at 35-37C with the production of both acid and gas. They form characteristic purplish red colonies in crystal violet neutral red Bile Lactose Agar (VRBL-Oxoid CM107). These colonies have a diameter of at least 0.5mm surrounded by a reddish zone of precipitation .This Procedure covers the use of the test for various food products.

Some coliforms do not exhibit the characteristics described above and this have be too confirmed by sub culturing into brilliant green broth. Confirmation for the presence of E.coli can be demonstrated by the indole test and the ability of this organism to grow at 44oC. After counting the colonies on the Petri dishes, the calculation of the final count is as follows; N= C/[ V (n1 + 0.1n2) d] Where = sum of all colonies on all Petri dishes counted n1 = n2 = d= V= number of dishes in the first dilution counted number of dishes in the second dilution counted dilution for which the first counts were obtained volume applied to each dish
Example Volume applied 1 ml Dilution 1/100 (10-2) 278 and 290 colonies Dilution 1/1000 (10-3) 33 and 28 colonies N= (278+ 290 + 33 + 28)/ [1 x (2+ {0.1 x 2}) x0.01] = 629/0.022 = 28590 or 2.9 x 104 cfu/ml
See procedure below.
Procedure for the Enumeration of Coliform Organisms by the Pour Plate Method 1.Purpose This procedure describes enumeration of coliform organisms (E. coli, Citrobacter, Enterobacter or Klebsiellaspp.). Coliforms are indicators of external contamination. Coliform organisms are able to ferment lactose within

24-48 hrs at 30C -37C with the production of both acid and gas. They form characteristic purplish red colonies in violet red Bile Agar (VRBL-Oxoid CM107). These colonies have a diameter of at least 0.5mm surrounded by a reddish zone of precipitation 2.Scope This Procedure covers the use of the Test for enumeration of coliform organisms. 3.Responsibility The Senior Bacteriological Technician is responsible for the implementation of the procedure. 4.Requirements 1.1 Sterile Violet Red Bile Agar (Oxoid CM 107) 4.2 Water bath @ 44 o C oC used for tempering the medium after sterilization 4.3 9 ml of quarter strength Ringers Solution or Buffered Peptone Water in Universal Bottles autoclaved at 1210C for 15 minutes. This is the diluent. 4.4 Sterile Petri dishes 4.5 One, 1,000l automatic pipette 4.6 Sterile blue 1ml pipette tips 4.7 Incubator at 30o C 1oC 4.8 Tryptone water 4.9 Water bath @ 44 4.10 Vortex mixer 4.11 Wire loop 4.12 Bunsen burner 4.13 Brilliant Green Bile Broth.(BGBB)
5.Reference Documents: Enumeration of coliforms colony count at 30 oC ( 03.05.05) D 4, Standards Unit, Health Protection Agency, UK.
6.
Procedure:

6.1 6.2 Samples are prepared as in described in the procedure for sample preparation and dilution. Mix the sample thoroughly by shaking the sample container 25 times in 10 seconds over 30 cm arcs. The sample may be shaken mechanically. The interval between mixing and pipetting should not be more than 3 minutes. Transfer 1ml of milk into 9 ml of diluent and mix thoroughly, either mechanically or by a Vortex mixer. This is the Primary dilution. Transfer 1 ml from the Primary dilution aseptically using a fresh pipette tip to a further 9 ml of diluent and mix. This is the 10 -2 dilution. Further dilutions are made by transferring 1 ml of each successive dilution to a further 9 mls of diluent using a fresh sterile pipette tip for each transfer. Pipette 1 ml from each dilution using a sterile pipette tip onto a sterile Petri dish starting with the highest dilution prepared. 15 ml of VRBL agar at a maximum temperature of 45 Care poured into each Petri dish and the agar and sample are mixed as below Mix the plate thoroughly by moving the plate horizontally 5 times followed by using a circular motion in a clockwise direction 5 times. Then repeat using vertical motion 5 times followed by a circular anticlockwise rotation 5 times. When the initial medium has set, a further thin layer (approx 4 ml) of sterile VRBA is poured on the surface of the medium When the medium has solidified, the Petri dishes are inverted and placed in an incubator at 30oC for 24 2 hrs Count the characteristic colonies which are dark red with a diameter of at least 0.5mm. Colonies of coliform bacteria are counted, if necessary, by a counter or by manually marking the underside of the plate with a marker pen. Confirmatory test If uncharacteristic colonies are present , inoculate 5 such colonies ( or all colonies if less than 5 present ) into (BGBB), including a representative of each different type of colony. Incubate BGBB tubes at 37 oC for 24 2 hrs. Consider colonies which produce gas in the Durham tube as confirmed coliforms.
6.3 6.4
6.5 6.6 6.8
6.9 6.10 6.11
7.0

For confirmation of E.coli inoculate 5 colonies into tryptone water, incubate at 44 oC for 24 2 hrs and test for indole production. Consider colonies that are Indole Positive as confirmed E.coli. Expression of Results Calculate the number of cfu, N, per millilitre of sample as follows:N= C/[ V (n1 + 0.1n2) d] C= sum of all colonies on all Petri dishes counted n1 = n2 = d = V = Example Volume applied Dilution 1/100 (10-2) Dilution 1/1000 (10-3) 1 ml 278 and 290 colonies 33 and 28 colonies number of dishes in the first dilution counted number of dishes in the second dilution counted dilution for which the first counts were obtained volume applied to each dish
Where
N = (278+ 290 + 33 + 28)/[1 x (2+ {0.1 x 2}) x0.01] = 629/0.022 = 28590 or 2.9 x 104 cfu/ml
6.12
Record Result in the Laboratory Day Book
5.8 Yeast and Moulds counts

This is a procedure used to determine the level of contamination of food samples, Milk & Milk Products with fungi,ie, yeast and moulds. The procedure is used in the Food & Dairy Hygiene Laboratories. When certain environmental conditions prevail, moisture, temperature etc fungal contaminants in food grow and release toxic secondary metabolite. These substances are called mycotoxins. Aspergillusflavusand Aspergillusparasiticusgrowing in cereals produces aflatoxin which affects man and animals. Fungi also cause food spoilage. National bodies have specifications on various products and to establish whether a product passes or not, yeast and mould tests have to be carried out. Potato dextrose agar, sabouraud dextrose agar and yeast extract agar are examples of media that can be used for the enumeration of fungi. This media should have acid added onto them to depress the growth of bacteria. Procedure for enumeration of yeast and moulds 2. Purpose This procedure describes the enumeration of yeasts and mouldsin milk and dairy products. 3. Scope This document covers the use of the procedure for milk & milk products in the Bacteriology and Food & Dairy Hygiene Laboratories. 4. Responsibility The Bacteriology technician is responsible for the implementation of the procedure. 5. Requirements 4.1 4.2 4.2 4.3 Sterile Potato Dextrose Agar tempered at 450C 10C in a conical flask Sterile 10% Lactic acid Sterile Petri Dishes Incubator set at 30C 10C

4.4 Water bath set at 45C 10C 4.4 Automatic 1,000l pipette 4.5 Sterile 1ml pipette tips 4.6 9 ml quarter strength Ringers Solution in Universal bottles. 6. Reference Documents: KEBS.KS 05-11: Parts 1-4 1976 ( Confirmed, 1999)
7. Procedure: Prepare serial ten-fold dilutions of the sample homogenate in 9ml BPW up to the desired dilution using a sterile pipette tip for each dilution. 6.2 Pipette 1ml from each dilution onto sterile Petri dishes starting at the highest dilution with a fresh sterile 1ml pipette tip. 6.3 Remove tempered medium from water bath checking that it is not above 450C 6.4 Add 1ml of sterile 10% lactic acid for each 100 ml of medium mix by swirling just before pouring onto Petri dishes 6.5 Add 15ml of Potato Dextrose Agar to each plate 6.6 Mix immediately after pouring by 5 to and fro movements followed by 5 circular clockwise movements followed by 5 to and fro movements at right angles to the first set, followed by 5 circular anti-clockwise movements. 6.7 Allow to solidify. Invert and incubate plates in the incubator at 30 C for 5 days. 6.8 Count plates containing 10-150 colonies. If mainly yeasts are present, plates with 150 colonies are usually countable. 6.9 Report results in colony forming units/g or colony forming units /ml depending on the type of sample. 6.1
Expression of Results Calculate the number of cfu,N, per milliliter or gramme of follows:N= C/ [V (n1 + 0.1 n2) d]
sample as
Where = sum of all colonies on all Petri dishes n1 = number of dishes in the first dilution counted n2 = d= V= Example Volume applied Dilution 1/100 (10-2) Dilution 1/1000 (10-3) 1 ml 278 and 290 colonies 33 and 28 colonies number of dishes in the second dilution counted dilution for which the first counts were obtained volume applied to each dish
counted
N = (278 + 290 + 33 + 28)/ [1 x (2 + [0.1 x 2]) x 0.01 = = 629/0.022 28590 or 2.9 x 104 cfu/ml calculation
Enter the results in the Laboratory Day Book showing the
Reporting of result Record the count expressed as two significant figures and expressed as a power of 10. When the third figure is less than five, do not change the preceding figure, when the third figure is 5 or more, increase the preceding figure by one unit. e.g 28,500 is expressed as 2.9 x 104 If the plate prepared from the 10 -1 .dilution contains less than 10 colonies, report the number of organisms as less than 1.0 x 10 2 per ml or g (derived from 10 x 1/d, where d the dilution is 10 -1 ). If there are only plates containing more than 150 colonies report the count as greater than 1.5 x 103per ml or per g. If all plates have uncountable colonies report as being an Estimated count . If sample is done undiluted, and no growth appears on plate, report as Nil CFU/ml.

7.0 Records Bacteriology Day Book
5. 9 Most Probable Number testing ( MPN) in water

Procedure for Most Probable Number Method (MPN)is used for detection and enumeration of coliforms organisms, thermotolerant coliforms organisms and presumptive Escherichia coli in water The purpose of this procedure is to enumerate coliform organisms in water using the MPN method. Coliforms organisms are used as indicator organisms. A water sample which has coliforms is most likely to have pathogenic organisms of faecal origin like Salmonella and Shigella sp. Coliform organisms ferment lactose in the testing media producing acid and gas. The gas production is indicated by the inverted Durham tubes. Fermentation is indicated by colour change like in MacConkey broth purple which changes from purple to yellow. E.coli is a coliform organism which is able to grow at 44 oC and breakdown trytophan with the production of indole.
Procedure for testing faecal coliforms and E.coli in water using the Most probable Number Technique. 1. Purpose This procedure ensures the detection and the enumeration of coliform organisms in water, thermotolerant coliform organisms and presumptive Escherichia coli by culture in a liquid medium in multiple tubes and calculation of their most probable numbers in the sample. It is applicable to all types of water. Coliform organisms are capable of aerobic growth at either 35 oC 0.5oC or 37oC 0.5oC in a liquid lactose culture medium with the production of acid and gas.Thermotolerant coliform organisms have the same fermentative properties as coliforms within 24 hr at either 44 oC 0.25oC or at 44.5oC. 0.25oC. E.coli is a thermotolerant coliform organism which also produces indole from tryptophan within 24 hr, at either 44o 0.25oC or 44.5oC. 0.25oC. 2. Scope This procedure is used in the Food Testing Laboratory. 3. Responsibility The Technicians is responsible for the correct implementation of the procedure.
4. Requirements Weighing balance (0. 01g) Spatula Autoclave Autoclave tape Bunsen burner Vortex Mixer Incubator @ 37oC 0.5oC Water bath @ 44oC 0.5oC Conical flask 1000 l automatic pipette Sterile 1 ml pipette tips Sterile 125ml bottles (one) Sterile 20mls Universal bottles (five) Water samples of at least 200mls Kovacs reagent for indole MacConkey Broth purple Durham tubes (big and small) Sterile tubes pH meter Tryptone water
5. Reference Documents: Water quality Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli Part 2: Multiple tube (most probable number ) method ISO 9308-2 6. Procedure: Test portions of the water sample are inoculated into a series of bottles and tubes as follows: Label the bottles and tubes (below) with the laboratory sample number Mix the sample of water thoroughly by inverting the bottle at least 10 times. Inoculate the bottles of the sterile MacConkey Broth purple as follows:

Add 50 ml of water sample to the bottle containing 50ml (double strength) of MacConkey broth Add 10 ml of water sample to each of five Universal bottles containing 10ml (double strength) of broth. Add 1 ml of water sample to each of five tubes containing 5 ml of (single strength) broth. Note Each bottle or tube must contain an inverted Durham tube for the collection of gas. Small Durham tubes are used for the tubes and medium sized Durham tubes are used for medical flat bottles. Mix the contents of each bottle or tube. Incubate the inoculated broths in a water bath at 44 oC for 24-48 hours with stoppers and caps loose. 6.2 Examination of the bottles/tubes Examine the bottles/tubes cultures after incubation for 18-24 hrs and regard as positive reactions those which show turbidity due to bacterial growth and gas formation in the Durham tubes, together with acid production (indicated by change of broth colour from purple to yellow).Reincubate those tubes which do not show any or all of these changes and examine them again for positive reactions after 48hr.
6.3 Confirmatory Test To confirm the presence of Presumptive E.coli, incubate a tube of tryptonewater,and test for indole formation after incubation at 44 oC for 24 hr by add 0.2-0.3 ml of Kovacs reagent to the tryptone water tube, the development of a red colour after gentle agitation denotes the presence of indole . 7.0 Expression of results From the number of tubes of isolation medium and confirmatory tests giving positive reactions, calculate by reference to the statistical tables below, the most probable numbers of coliform organisms, thermo tolerant coliform organisms and presumptive E.coli in 100ml of the sample. For example if a sample gives the following results; 50ml bottle positive(i.e gas and fermentation ), 3 bottles of 10 ml positive and 3 bottles of 5 ml positive

the profile will be 1 3 3. Using the table below this interprets as 18 faecal coliforms/ 100ml water. MPN values per 100 ml of sample and 95 % confidence limits (When one 50 ml, five 10 ml and five 1 ml portions are used) Number of reaction 1 of 50 ml 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 tubes giving positive MPN (per ml) 11 14 18 21 13 17 22 28 35 43 24 35 54 92 161 >180 95% confidence 100 limits Lower Upper 3 4 5 6 4 5 7 9 12 15 8 12 18 27 3 _ 26 34 53 66 31 47 69 85 101 117 75 101 138 217 450 _
5 of 10 ml 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5
5 of 1 ml 1 2 3 4 0 1 2 3 4 5 0 1 2 3 4 5
5-10. Detection of Salmonella sp

Members of the genus Salmonella are infectious pathogens capable of causing food poisoning and clinical symptoms in humans. They reach food directly or indirectly from

animal excreta at time of slaughter, from human excreta, water polluted by sewage and in the kitchen by transfer from raw to cooked food by hands or utensils. The genus is made up of a large group, which causes enteric fever; the main Salmonella sp that cause fever are S. typhi and S. paratyphi which causes typhoid and paratyhoidrespectively. Both are endemic in many developing countries. Most Salmonellae are found in the intestines of animals of pig, cows, goats, sheep, rodents and poultry. However S. typhi, and S.paratyphi are usually found only in humans. The two are excreted in the urine and faeces of infected individuals. Food poisoning with Salmonella sp and other bacteria is characterized by fever, headache, and diarrhoea and vomiting. Salmonella is usually present in much lower numbers in food. The organisms in food have been subjected to processing and the surviving organisms are often injured. So the method of Salmonella detection involves several stages to give the organism every chance to grow. The stages are; 2. Pre-enrichment in Buffered Peptone Water. 3. Selective enrichment in two broths. 4. Subculturing the broths onto two selective agar plates. 5. Identification of the bacteria using serological and biochemical tests, followed by API 20E. The different stages would be discussed as described in the procedure. See procedure below. Procedure for the Detection of Salmonella sp. in All Foods, Milk, and Dairy Products & Environmental swabs.
1.Purpose The purpose of this procedure is to determine whether Salmonella sp. is present in foods, environmental swabs, milk and dairy products. 2.Scope This procedure is used in Bacteriology for detection of Salmonellasp in all food types including milk and dairy products. .
3.Responsibility The Laboratory Head is responsible for the correct implementation of the procedure.
4.Requirements Weighing balance 0.01g Spatula Autoclave Autoclave tape Bunsen burner Vortex Mixer Incubator @ 37oC 1oC Water bath @ 45oC 1oC Water bath @ 41.5oC 1oC Hot plate Conical flask 1000 l automatic pipette Sterile 1 ml pipette tips Buffered Peptone Water (BPW), Selenite CystineBroth , Rappaport-Vassiliadis Soya Peptone Broth, Brilliant Green Agar (BGA) Xylose Lysine Deoxycholate Agar (XLD) Triple sugar iron agar slope Urea agar slope MacConkey agar Nutrient agar Salmonella polyvalent 'O' and 'H' antisera Wire loop 5.Reference Documents: Detection of Salmonella Species (16/09/2005) Protection Agency, Standard Units, UK
Reference no: F 13i2 Health

Procedure 6.1: Following the procedure described in the Procedure for preparation of samples and dilutions prepare 10-1 homogenate of the sample in buffered peptone Water (BPW). 6.2 Pre-enrichment Place the homogenate or swab suspension in an incubator at 37C 1oC for 18hr 2 h. For dehydrated foods the incubation period should be extended to 24hr 2 h 6.3 Selective enrichment:Transfer 1ml of the pre-enrichment buffered peptone water to 10ml of MKTTn broth incubates at 37C 1oC for 24 - 48hrs 3 h. Transfer 0.1ml of the pre-enrichment culture to 10ml of Rappaport-Vassiliadis Soya Peptone Broth (RVS) and incubate at 41.5C 1C for 24 3 hours. Subculture two loopfuls from each of the broths by streaking onto plates of brilliant green agar and xylose lysine deoxycholate agar. Place in an incubator at 37o 1C for 24hrs 3h. It is advisable to retain the incubated BPW under refrigeration until investigations are complete. 6.4 Recognition of colonies. On XLD Salmonella sp. colonies appears as red or red colonies with black centers. Isolated colonies may appear yellow with black centers. Lactose fermenting organisms may also appear as yellow with or without black centers. On BGA Salmonella sp. are red colonies surrounded by a bright red medium. 7.0 Confirmatory Tests 7.1 Typical (red or red colonies with black centers on XLD or red colonies surrounded by a bright red medium on BGA) or suspect colonies of Salmonella from each plate must be subjected to serological and biochemical confirmation. 7.2 Select at least five suspect Salmonella colonies including one from each plate of the selective agar and inoculate purity plates by sub culturing onto MacConkey agar. Incubate at 370C for 21hrs 3h Screen discrete colonies from the MacConkey agar biochemically using TSI (Triple sugar iron) agar slopes and urea agar. TSI needs streaking on the

surface of the slope and stabbing of the butt. Inoculation for the urea agar is the same as for the TSI agar. Incubate all media at 37 0C for 21 3h. Salmonella typically produce an acid (Yellow) butt with gas bubbles and an alkaline (deep pink) slope, with blackening due to hydrogen sulphide production. This blackening may mask the acid production in the butt, but is occasionally absent. Strains of Salmonella do not produce urease (rare exceptions) so no change in color is seen in urea agar. If biochemical results exclude the presence of Salmonella and the strain is pure no further action is required. Identify at least one isolate giving biochemical and/or serological reactions consistent with Salmonella with API 20E. Make sure that the API reagents are up to date and stored as per the manufacturers instructions. 8.0 Serological confirmation Subculture non-lactose fermenting colonies from the MacConkey agar to nutrient agar (NA) slope. Ensure that some water of condensation is present at the base of the NA slope, if none is present then add a few drops of sterile water. Inoculate the colony into the water of condensation and streak up the slope. Incubate at 370C overnight. Using the growth from the NA slope prepare three saline suspension on a slide using a loopful of saline and growth from the slope for 'O' antigens, the water of condensation at the bottom of the slope for 'H' antigens and a mixture from slope and condensate for auto agglutination. If auto agglutination occurs proceed to biochemical confirmation. Add a loop full of polyvalent 'O' and polyvalent 'H' antisera to two separate saline suspensions and rock the slide gently for 30 seconds. If agglutination occurs with the polyvalent antisera but not with the saline the reactions are considered to be positive. Final confirmation is done with the API 20E system. REPORTING OF RESULTS If Salmonella species are not isolated report as follows: Salmonella species not detected in 25g, 25ml or swab as appropriate.

If the isolate is confirmed as Salmonella species report as follows: Salmonella species detected in 25g, 25ml, or swab. The actual weight or volume of sample examined must be reported as, for example, 10g or ml, 25g or ml, 100g or ml. 7.0Records Bacteriology Laboratory Day Book
5.11 Enumeration of Staphylococcus aureus.

The purpose of this procedure is to isolate and enumerate Staphylococcus aureus from foods, milk and dairy products. Staphylococci are Gram-positive spherical bacteria that occur in microscopic clusters resembling grapes. Staphylococci are facultative anaerobes that grow by anaerobic respiration or by fermentation that yields principally lactic acid. Staphylococcus aureus forms a fairly large yellow colony on rich medium. The bacteria are catalase positive and oxidase- negative. Staphylococcus aureus grows at a temperature range 15 oC to 45oC degrees and at sodium chloride concentrations as high as 15 per cent Characteristics of Staphylococcusaureus Gram positive, cluster- forming coccus Non motile, non-spore forming facultative anaerobe Ferments glucose producing lactic acid Catalase positive Coagulase positive Dnase- positive Golden yellow colony on agar, usually haemolytic on blood agar, it produces the enzymes coagulase, DNase and catalase which are used to identify it.
Normal flora of humans found on nasal passages, skin and mucous membranes. Pathogen of humans, causes a wide range of suppurative infections, as well as food poisoning and toxic shock syndrome.

Staphylococcus aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of pyrogenic exotoxins into the blood stream. In processed foods Staphylococcus aureus is readily destroyed by heating, drying, and other processing conditions to which the food is subjected. Thus the presence of Staphylococcus aureus indicates contamination from the skin, mouth, or nose of food handlers. Contamination of processed foods may also occur when contaminated food collects on processing surfaces to which food products are exposed. Large number of Staphylococcus aureus cells in processed foods indicates that sanitiation, temperature control or both were inadequate. This finding, however, is not sufficient evidence to incriminate a food as the cause of food poisoning. The isolated Staphylococcus aureus organisms must be shown to produce enterotoxins. In the Staphylococcus group of organisms, only Staphylococcus Staphylococcus epidermidis are significant in their interactions with humans. aureusand
Screening for Staphylococcus aureus in foods is done using a selective, Baird Parker Medium where it grows as shiny black colonies. Further confirmation is done using the enzymatic tests mentioned above; catalase,coagulaseand Dnase. See procedure below. Procedure for the enumeration of Staphylococcus aureus 1.Purpose The purpose of this procedure is to isolate and enumerate Staphylococcus aureus from foods, milk and dairy products. 2.Scope This procedure applies to Bacteriology Laboratories. 3.Responsibility The Senior Bacteriology Technician is responsible for the implementation of the procedure. 4.Requirements Spatula Bunsen burner Vortex Mixer

Water bath @ 45oC 1oC 9ml sterile quarter strength Ringers solution in Universal bottles autoclaved @ 121oC for 15 minutes Sterile Petri dishes Mechanical blender Automatic 1ml pipette Sterile 1 ml pipette tips Balance capable of weighing to 0.01g Incubator at 37oC 1oC Buffered Peptone Water (BPW) Baird Parker (BP) medium Sterile spoons Sterile 1 ml and 10 ml glass pipettes graduated in 0.1ml volumes Sterile spreading rods DNase medium Staphylase Test Reagent Kit (Oxoid,UK)
5.Reference Documents: Enumeration of Staphylococcus aureus, Reference no F 12, Health Protection Agency, Standard Units, UK, 03.05.05 6.Procedure: 6.1 Following the procedure described in Procedure for preparation of samples and dilutions, prepare 10-1 homogenate in buffered peptone water (BPW) and further decimal dilutions as required. 6.2 Starting with highest dilution to be plated, aseptically transfer 0.5ml of a sample of each dilution suspension onto its own Baird Parker plate. Spread inoculum over surface of the agar plate using a sterile bent glass streaking rods and let the plates dry. 6.3 Invert the plates and incubate for 48 2 hours at 37 oC 1oC. 6.4 Countand record colonies. Examine the plates for typical colonies of Staphylococcus aureuson plates containing up to 150 colonies. Typical colonies appear as black, shiny, convex colonies up to 3mm in diameter, with a narrow zone of opacity surrounded by a zone of clearing. Count and record the number of typical colonies. For foods

of bovine origin, including dairy products, atypical colonies of Staph.aureus may occur but do not show opacity or clearing. For foods of this type also count and record atypical colonies. 6.5 Confirmatory tests Sub culture five colonies of each type for (or all colonies if less than five) for confirmatory testing using Dnase and coagulase production. 6.6 Inoculate each colony onto a DNase agar plate and plate out onto a segment of a blood agar plate. Set up blood agar plates and DNase agar plates with a positive control strain of Staphylococcus aureus and a negative control strain of Staphylococcus epidermidis to verify performance and incubate alongside the plates of the test organism. Transfer the plates to an incubator at 37oC 1oC for 18-24 hours. 6.7 Examine the blood agar plates for purity and colonial morphology consistent with S. aureus cream or golden coloured colonies up to 3mm in diameter.
6.7 DNase production Flood the DNase plates with normal hydrochloric acid (HCL). After about 30 seconds, discard the excess reagent (HCL) into a chemical waste container. Positive reaction occurs when colonies show a defined zone of clearing. 6.8 Coagulase production Using the growth on blood agars (BA), perform a slide agglutination test on the strains giving a positive DNase test. Compare the results with the growth from the Blood agar plates of the control Staphylococcus aureus and Staphylococcus epidermidis.
7.0Control cultures Positive and negative controls must be used for confirmatory tests. A positive reaction shows clumping within ten seconds and a negative reaction shows no clumping within ten seconds and thus no coagulase produced. Positive control S. aureus (Oxford strain) NCTC 6571

Negative control S. epidermidisNCTC 11047 Record the results of the control strains in the day book. 8.0 Calculations Counts should be calculated where possible using dilutions giving 15 or more colonies on the plate. Calculate the count of Staph. aureus as follows:Counts of Staph.aureus per gram= Number of typical colonies confirmed x Number of colonies counted Number of colonies tested Volume tested x dilution
Added to;Number of atypical colonies confirmed x Number of colonies counted Number of atypical colonies tested Volume tested x dilution
9.0 Reporting of results If no colonies of the test organism are present on the 10 -1 dilution, report as Less than 20 cfu /g or ml. This indicates a LOD less than 20cfu. If the test organism is detected with counts between 20 and 99 per gram report in the form of: acfu/g or ml ( Where a is a number between 20 and 99)
If the test organisms are detected at counts of 100 or higher per gram, report with one figure before and one figure after the decimal point expressed to the power of 10 in the form of : ax 10bcfu/g or ml
(Where a is never less than 1.0 or greater than 9.9 and b represents the appropriate power of ten. Round counts up if the last figure is 5 or more and down if the last figure is 4 or less: e.g 1920 cfu/g or ml is reported 3 as 1.9 x 10 cfu/g or ml 235,000 cfu/g or ml is reported as 2.4 x 10 5cfu/g or ml Plates with more than 300 colonies. When number of CFU per plate exceeds 300, for all dilutions, record the counts as too numerous to count or uncountable (TNTC or UC) for all plates. Mark calculated count as estimated to denote that it was estimated from counts greater than 300 per plate. If all plates have uncountable colonies report as being an Estimated count. Based on the highest dilution measured. 7.10 Records Laboratory Day Book.
5.12 Acidity Test - Titratable Acidity of Milk

Introduction: The test is performed to determine whether the raw milk is stable to heat-treatment. This is meant to measure the level of total acidity in milk and reported as lactic acid (LA). The apparent acidity of fresh milk is a property and components of milk which falls between 0.12 - 0.16% LA. The development of acidity is due to activities of microorganisms present in the milk. Acid development in milk affects the pH and its stability to heating. Milk clots on boiling when its acidity is about 0.20% and clots at room temperature when the acidity is about 0.5% lactic acid. This test is carried out by titration and is based upon the chemical principle that equal volumes of acids and alkaline of the same strength will exactly neutralize each other.

The point of neutralization is determined by means of an indicator which gives one definite colour in an alkaline medium and another colour in an acid medium.
Procedure for titratable Acidity of milk. 1. References. KS 05-30 (2001) KS 05-34 2.
Specifications for Pasteurised milk Specifications for yoghurt
Requirements Standardised N/9 NaOH 10ml Pipettes and Pipette filler 2 Beakers (50 or 100ml) Burette 10ml (with identity number and validated) Phenolphthalein Indicator (2.5%) freshly and not exceeding 6 months since preparation Thermometer range (-100C to +1100C)
3.
Procedure. 3.1 Mix each sample well and adjust the temperature of the milk if necessary to 20 0C 1.00C. 3.2 Fill the burette with standardised N/9 NaOH.
3.3 Remove any excess NaOH from the tip of the burette with a tissue and adjust the volume to a convenient starting reading 3.4 Pipette 10 ml of the sample into a beaker. 3.5 Add 2-3 drops of phenolphthalein indicator to the milk in the beaker and agitate by rotating the contents. 3.6 Note the initial volume of N/9 NaOH in the burette when starting to titrate 3.7 Titrate the sample quickly and continuously by adding the N/9 NaOH from the burette into the beaker until the first permanent faint pink colour appears and

persists in the whole volume of the milk for at least 10 seconds, stop adding NaOH and note the volume N/9 NaOH in the burette at the end (final). 3.8 Calculate the volume of NaOH used by subtracting the initial from final volume shown on the burette used. The volume of NaOH used divided by 10 gives the percentage acidity of the sample and this is expressed as % lactic acid (LA). Example; Volume of NaOH solution used = 1.5ml, Hence milk acidity shall be 1.5/10 = 0.15%LA The natural acidity in milk is due to the presence of phosphates, calcium and carbon dioxide. Developed Acidity is due to microbial growth. 7. Results Standards. Fresh milk acidity range is (0.13 0.15%) and Cream (0.08 0.11%) while the acidity of fermented product depends on stage of fermentation. 9. RecordsLaboratory day book
5.13 Butterfat Fat content Gerber Method

Rapid volumetric methods are often used for routine purposes for determing fat in milk, Gerber method is commonly used. This test method gives value for fat content in grams of fat per 100g of milk. Principle separation of the fat of milk in a butyrometer by centrifuging after dissolving the protein with sulphuric acid, the separation being aided by the addition of a small quantity of amyl alcohol. The butyrometer is graduated to give a direct of fat content. The butter fat test of the same milk was compared with results generated by lacto scan milk analyser, Procedure for the Determination of Butter Fat in milk using the Gerber Method 1. Purpose This procedure ensures that the butter fat analysis of raw and pasteurized milk is properly conducted.

2. Scope This test is only used in milk, raw or pasteurized regardless from which animal. Responsibility The Head of the Food and Milk Hygiene Laboratory is responsible for the proper implementation of this test. Reference Documents KS 05-12: Parts 1-2: (1976) Confirmed (1999) Determination of Fat Content in Milk. Requirements: Sulphuric acid (H2SO4) GP or Analar Grade with a specific gravity of 1.815 0.002g/ml at 200C or 90% Concentration of H2SO4 Amyl alcohol, Analar Grade with a specific gravity of 0.809 - 0.813 at 20 0C 10.94 ml Gerber pipette (with ID and validated) 1ml Dispenser or pipette (numbered and validated) 10ml aciddispenser (numbered and validated) Milk butyrometers range (0- 8%) (with a numbered butyrometer) Double ended stoppers for butyrometers Water baths at 400C 1.00C and 650C 1.00C. Gerber Centrifuge able to attain 1,100rpm Thermometer with range -100C to + 110 0C that is internally calibrated and engraved for identification Timer with ID Pipette filler Rack for shaking butyrometers Goggles for eye protection
4.
5.
6.
Procedure: 6.1 6.2 Warm the milk sample in a water bath to 40 0C 1.00C, mix it well and then cool to 20 0C 1.00C Dispense 10 ml of Sulphuric Acid into each butyrometer.

6.3 Confirm the temperature, mix the milk and transfer 10.94 ml of milk using Gerber pipette into each butyrometer allowing it to flow slowly and gently down the side of the butyrometer to avoid burning of the milk by the sulphuric acid. Slowly add 1.0ml of amyl alcohol from a tilt bottle. Cork the butyrometers with stopper without disturbing the contents until half of the cork is into the butyrometer . Shake well in a shaker or by hand until a chocolate brown colour is attained. Centrifuge for 5 minutes at 1,100 rpm. Place the butyrometer in the water bath at 65 0C 1.00C for 3 minutes. Adjust the meniscus (by moving the stopper) formed by the junction of the milk and acid to the bottom of the butyrometer scale. Read off the upper level and lower level of the butter fat using the bottom of the meniscus from the butyrometer scale. The difference between the two readings gives the percentage butter fat in the milk by mass. IQC regime, test to be carried out in duplicate. Acceptable repeatability range for this test is 0.1, if the error exceed this range a repeat of test will be carried out and mean calculated from results of four tests.
6.4 6.5
6.6 6.7 6.8
6.9 6.10
5.14 Resazurin Test

This test is used to determine in a general way the bacteriological quality of raw milk and determine its keeping quality. Resazurin, which is added to milk in liquid form, is a redox indicator. When the oxygen potential of the milk is normal, the colour will be blue but if the potential is lowered because of metabolic activity by micro-organisms the colour will change to pink or even white. Resazurin reduction occurs in two stages, the first an irreversible change from the blue resazurin to the pink resorufin and the second a reversible change from the pink resorufin to the colourlessdihydroresorufin. This is a dye reduction test which is based on the ability of micro-organisms to alter the oxidation-reduction potential (the redox potential) if a medium which is reflected through a colour changes of the dye. False reduction can be assumed to be brought about by the leucocytes if the colour in the downgraded milk sample (for example, milk from animals suffering from mastitis) remains unchanged for a longer time than observed normally.
The solution of resazurin is prepared by adding one tablet to 50mls cold sterile distilled water. When the tablet is completely dissolved a 0.005% standard resazurin solution is obtained. When not in use the solution should be kept in a cool dark place preferably a refrigerator and be discarded when more than 8 hours old.
Disc reading 6 and 5 4 3&2 1& 0
Colour Deep & light blue Deep pink pink & pale pink slight pink & white
Keeping Quality Satisfactory Acceptable Reject Reject
Procedure for the Resazurin Test on Raw or Pasteurized Milk 1.Reference Documents: KS 10: 2006 Specification for Raw Whole Milk 2.Requirements Water Bath at 370C 1.00C Sterile Resazurin Test Tubes and sterile rubber stoppers Resazurin Solution freshly made 10ml graduated pipettes, numbered and validated 1ml pipette Freshly made Resazurin solution Lovibond Comparator with standard Resazurin disc, having seven standards numbered 0-6 (disc 4/9) Thermometer with range -100C to + 110 0C that is Analabs internally calibrated and coded for identification Timer Test tube rack for holding test-tubes. 50ml measuring cylinder 50 l of the milk sample to be tested 3.Reagent Sterile standard resazurin solution

4.Procedure: 1. Wash and clean the test tubes and rubber stoppers and rinse with distilled water. Sterilize in the autoclave at 121 0C for 15 minutes. Two tubes are required, one for the sample and the other for the test control. 2. Heat a portion of the test milk to boiling point and cool to room temperature. This is for the test control. 3. Mix the test sample and the control sample, separately, by inverting each at least 25 times. 4. Aseptically pipette 10 mls of the test sample into one test tube and add 1 ml of resazurin solution. Prepare the control tube in the same manner using boiled milk, stopper the tubes and invert three times. 5. Mix by inverting the tubes twice, place the tubes in the water bath at 37.50C 0.50C and note time. For Raw milk read and report after 10 minutes, while pasteurized milk read after 30min, 1, 2, and 3 hours. Report the results of third hour. 6. Place the tube containing the prepared sample in the right hand compartment (when viewed from the front of the instrument) and the control tube in the left hand side. 7. Use indirect light, match the sample with one of the comparator numbers. When the colour falls between two disc numbers, record the sample as the lower number value and add a half. 8. IQC regimes set the test in duplicate. 5.0 Interpretation of Results Colour of Sample White or complete reduction of pink colour (stop test) Pale Pink, pink & white mottling or pink band at the top with paler pink below Deep Pink Light Blue Deep Blue Control Sample 6.0 Records
Comparator No 0
Milk Grade Reject
1 or 2 3 or 4 5 or 5.5 6 6
Reject Reject 2nd Grade 1st Grade

Laboratory Day Book
5.15 Alcohol Test

The alcohol test is used for rapid assessment of stability of milk to processing. The test is useful as an indication of the mineral balance of milk and not so much as an index of developed acidity. The test aids in detecting abnormal milk such as colostrums, milk from animals in late lactation, milk from animals suffering from mastitis and milk in which mineral balance has been disturbed. The test is based on diffusion of water from milk to alcohol, this migration results in precipitation of milk. This only occurs when milk quality is questionable or its failing. The concentration of alcohol used is 70% or 75% Procedure for the alcohol Test in Raw Milk. 1. Purpose The purpose of this test is to determine the stability of milk proteins when equal parts of milk/alcohol are mixed. The test evaluates suitability of unprocessed milk for further processing or shelf-life of processed milk. Chemical instability in milk is due to colostrum, mastitis or high acidity.
2 Scope This procedure covers raw and pasteurized milk regardless of its source.
3 Responsibility. The senior Technician in Food & Dairy Hygiene is responsible for the proper implementation of this procedure.
4 References. KS 05 -10:1992 Specification for Unprocessed Whole Milk 5 Requirements


Alcohol Gun 70 % Ethanol in distilled water 6 Procedure. Fill alcohol gun with 70 % alcohol (Ethanol). Hold the alcohol gun almost at perpendicular to milk sample with the receiver cup up. Dip the tip of the gun into test milk, the open tip will sample approx. 1ml of milk Close the opening of receiver cup with the thumb Invert the gun, to allow milk and alcohol flow into the receiver cup. Shake the mixture for 30 sec and observe for any coagulation or precipitates .
7 Report Coagulation or precipitate Alcohol positive No coagulation Alcohol negative
Interpretation A negative test indicates low acidity and good heat stability of milk sample. Note any flakes or clots. The presence of a flake or a clot denotes a positive. Milk showing positive is not considered suitable for the processing, as it is unstable on heating.
5.16 Moisture Analysis in Foods

Principle Free water in foods or any other material evaporate on heating, hence change in weight. The principle is weight to weight basis The loss in weight is calculated as percentage of the original weight of sample. Moisture content is of great value in food science because it determines the rheological characteristic of food, moisture levels above set limits encourages growth of microorganisms especially moulds in dry foods Moisture content can be carried out by heating in oven at 105 0C for 4 hrs, cooled in desiccators and weighed.

Moisture analysis can also be done using gravimetric method; the machine combines heating and weighing simultaneously. Heating is by halogen lamp. For the accurate results the test requires sample weight of at least 5g, After preparing and setting the machine, start the test and allow time until the test ends. Allow the machine to cool to near room temperature before weighing second and third repeat tests. The cooling lowers rate of evaporation during weighing and spreading of the sample which results to a lower moisture content of the material at the end of the test. When the test is completed the machine beeps ones and the LCD display test over, results of the test M/ content as percentage and time taken.
Procedure for the Determination of Moisture content by Ohaus MB45 Thermo gravimetric Method 1. Purpose This procedure ensures that moisture in dairy products, artemisia and other food samples is correctly determined using the Ohaus Moisture Analyzer MB45 model. 2. Scope This instruction covers all types of food and feed samples, dairy products (butters, cheese, ghee, milk powder) and all dried food samples. 3. Responsibility The Head of the laboratory is responsible for the correct implementation of this procedure. 4. Reference Documents: Instruction Manual MB45 Moisture Analyzer.Certified ISO 9001 QMS.Ohaus Corp. New Jersey. USA 5. Requirements MB45 Moisture Analyzer machine Sample pan, Spatula or spoon

Sample Standard weight 5g OTR Form 6. Procedure 6.1 Switch on the Moisture Analyzer 6.2 Allow the machine to warm-up for about 30 minutes 6.3 Open the cover on the Moisture Analyzer, place the empty pan in the panhandle and place in the sample chamber and tare the weight 6.4 Place a 5 gram standard weight on the pan and confirm and record the weight. 6.5 Program the machine to Identify the sample either by - Name or Lab No. Drying temperature - 1050C Temperature program - Standard Switch of criteria 6.7 Tare the pan 6.8 Weigh approximately 5g of sample onto the pan. 6.9 Close the cover of the Moisture Analyzer 6.10 Press start button to start the process. 6.11 Monitor the progress of the machine until Test Over is displayed on LCD 6.12 Record the result as percentage moisture
5.17 Phosphatase Test for Pasteurized Milk

Pasteurization Heat treatment process applied to liquid milk the objective of eliminating possible health hazards arising from pathogenic micro-organisms associated with milk. This can be achieved through a) Batch method Milk is heated to 650C and maintained at this temperature for at least 30 minutes and immediately and rapidly cooled to 100C or less.

b) High temperature short time method (HTST) Milk is heated to 730C and maintained at this temperature for at least 16 seconds and immediately and rapidly cooled to 100C or less. c) Flash pasteurization Milk is heated to 800C and maintained at this temperature for at least 10 seconds and immediately and rapidly cooled to 100C or less. The principle on which the test is based is that although Phosphatase enzymes are invariably present in raw milk. They are inactivated during pasteurization. It has been shown that this enzyme is more difficult to destroy that the most heat-resistant pathogenic organisms which are likely to be present in milk. The test involves incubation of the milk with disodium p-nitrophenyl phosphate under alkaline conditions. If the milk contains Phosphatase a yellow colour is produced due to the formation of p-nitrophenol. The degree of destruction of the Phosphatase in the milk during pasteurization is then assessed by comparing the colour produced with the standard colours on a comparator disc. Procedure for Phosphatase testing in pasteurized milk. 1.Purpose To determine whether the milk is properly pasteurized 2.Scope This test covers pasteurized milk only. 3.Responsibility The Senior Technician of the laboratory is responsible for the correct use of this test 4.Requirements Sterile test tubes and well-fitting rubber stoppers 1, 5, and 10 ml pipettes with ID and validated Phosphatase Reagent (4.nitrophenol phosphate disodium salt) Lovibond comparator with phosphatase disc 4/13 Incubator 370C 1.00C Sample of pasteurized milk to be tested
5.Reference Documents: David Pearson 1970: The Chemical Analysis of Foods 6th Ed pp 437-439. 6.Procedure: 6.1 Transfer 5mls of the Phosphatase reagent solution to a test tube using a pipette and 1ml of the milk to be tested, stopper the tube and mix the content well by invert 3 or 4 times. Label the tubes and incubate at 370C 1.00C for exactly 2hrs. Run controls using ultra heated milk or freshly boiled milk of the same sample and incubate at the same time together with the samples. Take the reading using the comparator. The blank shall be on the left hand slot of the comparator and the sample on the right hand side. Revolve the disc until the test sample is matched. Prepare fresh Phosphatase solution if it has turned yellow, other wish renew the solution after every 3 weeks
6.2 6.3 6.4 6.5 6.6
Interpretation Comparative readings 0.10 = Properly pasteurized 11-18 = slightly under pasteurized 19- 42 = under pasteurized >42 = grossly under pasteurized
5.18 Antibiotic Residue in milk and Meat/Liver

Introduction The Antibiotic ampoule is composed by a gel matrix containing Bacillus stearothermophilusvar. calidolactis spores, nutrients and a pH indicator (bromocresol purple). B. stearothermophilus spores are very stable at room temperature and need nutrients and heat to germinate and grow in its vegetative form.

When B. stearothermophilus grows, it consumes the sugar present in the medium, releasing acid as a metabolite. The consequent drop of pH is revealed by the change of colour of the pH indicator, from purple to yellow. When adding a sample of milk on the agar surface, it diffuses into the media, carrying all the substances dissolved in the milk. When the system is incubated at the proper temperature, the B. stearotermophilus spores will germinate and multiplicate, acidifying the agar medium and changing its colour, unless an inhibitory compound is present in milk sample in an amount able to inhibit vegetative cells growth. By varying the parameters of the test performance (spores concentration, nutrients, incubation temperature and time, amount of milk added, etc.) it is possible to set up the colour change in order to detect fixed amounts of selected inhibitory substances. A threshold of sensitivity for these substances is characteristic of the test, and milk containing one of those substances in a concentration higher than that threshold will inhibit the colour change of the test in standard conditions. Procedure for the Determination of Antibiotics in Fresh Milk 1. Purpose This ensures that the correct procedure for determining presence of antibacterial substances in milk is carried out. The result of the test indicates presence or absence of antibiotic residues. 2. Scope This procedure covers Raw and Pasteurized milk from any source (Cow, Goat or Camel) Note the test is extremely sensitive to antibacterial substances (antibiotics, sulpha compounds and other components such as detergents, disinfectants) 3. Responsibility Technician in charge 4. References DelvotestR SP-NT/ SP MINI-NT User instruction guide 5. Requirements DelvotestR SP NT ampoule with solid purple agar medium

Syringe and Disposable pipette tip supplied with kit A pair of scissors Incubator or water bath at 640C 10C Timer
6 Procedure 6.1 Pre heat the incubator or water bath to 640C 10C 6.2 Cut off ampoule from the block; take care not to damage the foil from adjacent ampoules. 6.3 Open the ampoule by punching a small hole in the aluminum foil, but do not remove foil from ampoule. 6.4 Attach firmly a disposable pipette tip onto the syringe without touching tips. 6.5 Pipette out 0.1ml of milk sample by depressing the plunger of the syringe completely and release when the tip is in milk. 6.6 Transfer the milk sample in the pipette (0.1ml) completely into an ampoule. By depressing the plunger slowly adding the milk directly onto the agar medium. 6.7 Place ampoule with sample in a preheated incubator or water bath 64 0C 0 1 C and incubate for 3 hours. 6.8 IQC regimes; carry out the test in duplicate per batch (12 samples) i.e. for every 12 samples do one sample in duplicate, This is a detection test and has only two options positive or negative.
7 Reading After 3 hours incubation withdraw the ampoule and read 7.1 Yellow colour indicates the absence of antibacterial substances in a concentration at or below the test's detection level 7.2 Yellow / purple colour indicates the presence of antibacterial substances in the related milk sample close to the test's detection limit. 7.3 Purple colour indicates the presence of antibacterial substances in the related milk sample at or above the test's detection limit.
5.18.Chlorine Content in Water

Chlorine content in waster should be determined as soon as sample arrives in the lab, and where possible at a point of sampling. The training will highlight the determination of Free Residual and Total Residual chlorine. Training to cover how to calculate combined chlorine This procedure ensures that water samples are correctly assessed for its chlorine content and suitability for use. Procedure for assessment of water for chlorine content. 1. Requirements Lovibond Chorine (DPD) Checkit Lovibond test DPD tablets Nos 1 and 3. 2. Procedure 6.1 6.2
6.4
Rinse the comparator cell with the sample water. Pour enough sample water to cover the tablet and add one DPD No. 1 tablet. Shake and allow to disintegrate and then make the volume up to 10 ml and Mix and Match the colour of the water in the cell with the one on the comparator and record. This is the residual chlorine. For total residual chlorine add one DPD 3 tablet to the same cell. Mix and match at once and take the reading of the comparator disk. The combined residual chlorine is the difference between the free residual chlorine and the total residual chlorine.
Note: Chlorine added to water reacts first with hydrogen sulphide and organic matter until these are neutralized. The remaining chlorine is the amount available to destroy remaining micro-organisms. Standard: Free residual chlorine of 0.3 - 0.5 ppm is recommended for both process and drinking water. 3 Records Laboratory Day Book
5.19 0Brix
Concentrations of sucrose and other sugars in fermented products can be determined by several methods (Refractometry, spectrophotometry and chemical titration) In refractometry a drop of sugar solution is placed on to standardized refractometer prism and viewed against a source of light. The results can be read on LCD as 0Brix, which directly relates to sugar concentration. Monosaccharide and refractmeter methods disaccharide sugars concentration can be determined by
In fermented milk products the most common sugars are disaccharide (lactose and sucrose) Lactose is normally the milk sugars, its content can be determined by analyzing the Brix of natural yoghurt and lala, while sucrose are commonly added as sweeteners in sweetened products. Procedure: Wet the prism pot of refractometer with distilled water Clean the prism of the refractometer using lens tissue. Be careful not to scratch the prism. Switch on the refractometer Apply a drop of test sample on to the prism pot Brix, Press read to check for sugar concentration. The reading on LCD display is the results of sugar concentration as 0Brix. Wash off the sample from the prism with distilled water and then wipe the prism dry with soft tissue. Average the three results and record in the Day Book as 0Brix.
0
Brix for sweetened products is a combined concentration of lactose and sucrose.
5.20 Procedure for Determination of Hydrogen Peroxide in milk

1Purpose This instruction ensures that levels of hydrogen peroxide in milk are accurately determined. 2. Scope This procedure covers the determination of hydrogen peroxide in raw milk and pasteurised milk using hydrogen peroxide strips.
3. Responsibility The senior Technician is responsible for the proper implementation of this procedure.
4. References. Quantofix R User Manual Operating Kit.
5. Requirements Test Sticks QUANTOFIXR Peroxide 25 Kit.
6. Procedure. Ensure that the test strips are not expired. Remove test sticks as required and reseal the container immediately. Avoid touching the test paper zone. Dip test stick about 2 sec into the test milk Shake off excess liquid and read off after 15 sec Compare the test paper zone with the blue colour scale on the Quantofix R Peroxide 25 sticks can to determine the concentration of Hydrogen peroxide in the milk. In the presence of hydrogen peroxide, the test paper turns blue the intensity of the colour depends on amount of hydrogen peroxide in the milk sample.

Report as mg/l, Hydrogen peroxide (H2O2)
7.0 Storage Avoid exposing the test sticks to sunlight and moisture; store the pack in refrigerated conditions (2 to 80C).
5.21 Procedure for the Sediment test for Raw and Pasteurized Milk
1Purpose The purpose of this test is to grade milk quality based on the level of dirt in fresh raw or pasteurized milk. 2. Responsibility This procedure is used in the Food Testing Laboratory. 3. Reference Documents: 4. KS 05 -10:1992 Specification for Unprocessed Whole Milk 5. KS DKS 05-30 2001 Specifications for Raw Milk 6. Requirements Gerber Sediment tester attached to Vacuum Pump with a suction pressure of 5mmHg Thermometer range - 100 to+1100C Sediment test cards. Water Bath at 400 1.00C Filtered or distilled water Standard grading card (Gerber Original) litre of the Raw Milk to be tested for Sediment 7. Procedure: Rinse the sediment tester with filtered or distilled water. Place a clean filter pad in the base of the sediment tester. Take 1/2 litre of the Raw Milk to be tested. Agitate well

Warm the milk sample in the water bath to 40 0C 1.00C.Mix well by agitation and cool to 200C 1.00 Connect the sediment funnel to the pump, switch the pump on and pour the 1/2 litre of milk through the sediment tester funnel. Remove the filter pad and place on absorbant paper to dry for at least 15 min at room temperature. Grade the amount of dirt against the Standard Grading card and record the results. 9. Evaluation of Result Compare the Sediment test cards after filter and drying with Standard Grading card and ward the grade of the milk accordingly .

ANNEX 1 Procedure for preparation of Quarter Strength Ringers solution 1.Purpose This procedure ensures the proper preparation of quarter strength Ringers solution that is used as a diluent in various procedures. 2.Scope This covers the Bacteriology Laboratories. 3.Responsibility The Senior Bacteriology Technician implementation of the procedure.
is
responsible
for
the
proper
4.Requirements Full Strength Ringers Solution. (See 6.1 below) 500 ml volumetric flask with identification number Sodium chloride GPR Potassium chloride GPR Calcium chloride anhydrous GPR Calibrated Thermometer -100C to +1100C Sodium bicarbonate GPR pH meter distilled water Weighing balance (0.01g) 6. Reference Documents: Cheesbrough M (2000) District Laboratory Practice in Tropical Countries Part 2 pp 445
7. Procedure: 6.1 Using the full strength formula (or tablets, prepare 500 ml Full Strength Ringers as below: Sodium chloride 4.50gms Potassium chloride 0.21gms Calcium chloride anhydrous 0.24gms Sodium bicarbonate 0.10gms

Distilled water 6.2 500ml
Weigh accurately the dry ingredients and transfer to a clean dry 500ml volumetric flask.
6.3
Half fill the flask with distilled water, and mix until the chemicals are fully dissolved. Make up to 500ml mark with distilled water, and mix well. 6.4 Transfer to a clean bottle and label Full Strength Ringers Solution and mark with the date prepared and the operator and store at 2 8 C. 6.5 To make 1,000 ml of Quarter strength Ringers solution Full strength Ringer's Solution 250ml Distilled water 750ml Check that the pH is 6.9- 7.1 using a pH meter and if not adjust the pH. Dispense as required and sterilize by autoclaving at 121C for 15 minutes.

ANNEX 2 Making sterile blood agar plates 1. Purpose This procedure ensures that sterile Blood Agar medium plates for routine pathogen isolation are properly prepared. 2. Scope This procedure applies to the Bacteriology laboratory. 3. Responsibility The Bacteriology Technician is responsible for the proper implementation of the procedure. 4. Requirements Dehydrated Blood Agar base culture media (Oxoid No.CM0055) Distilled water Conical flask 500 ml Sterile Petri dishes Weighing boat Thermometer Autoclave Autoclave tape Weighing balance ( 0.01g ) Spatula Water bath at 45oC 1oC Sterile Defibrinated Sheep blood 500 ml Measuring Cylinder 5. Reference Documents: Oxoid Manual 7th Edition 1995 pp 2-48 6. Procedure. Weigh and suspend 20g Blood Agar base in 500mls of distilled water. Bring to the boil and dissolve completely. Autoclave at 121 for 15 minutes. Cool the base agar to 45C 1oC in a water bath. Add 5% defibrinated sheep blood, mix with gentle rotation and pour into sterile Petri dishes (about 20ml of the medium per Petri dish). Leave overnight to set on a flat bench.

Carry out Quality Control. Label the plates and store at 2-8C

ANNEX 3 Making McConkey agar 1. Purpose This procedure ensures that MacConkey Medium for routine pathogen isolation is properly prepared. 2. Scope This procedure covers the preparation of MacConkey media in a Bacteriology Laboratory. 3. Responsibility The Bacteriology Technician is responsible for ensuring that this procedure is correctly carried out. 4. Reference Documents: Oxoid Manual. 7th Edition 1995 pp 2-142 5. Requirements MacConkey Agar (CM0007) Distilled Water Petri Dishes One litreConical flask Weighing boat Thermometer Autoclave Autoclave tape Weighing balance 0.01g Water bath at 45C 1oC Spatula pH meter 6. Procedure Weigh and add 52 g MacConkey Agar medium to 1litre of distilled water. Bring to the boil to dissolve completely. Autoclave the media at 121C for 15 minutes. Cool to 45 0C in water bath. Media should not be left at 450C for longer than 3 hours. Pour into sterile Petri dishes (20 ml of medium per Petri dish) and leave to set. Carry out Internal Quality Control. Label store at 5oC 3oC

ANNEX 4 Performing the Catalase test 1. Purpose The purpose of the procedure is to differentiate bacteria that produce the enzyme catalase from non-catalase producing bacteria. It is a standard procedure to differentiate between Staphylococcussp. and Streptococcus sp. Scope This test is used in the Bacteriology laboratories. Responsibility The Senior Bacteriological Technician is responsible for the correct implementation of the procedure. Requirements Hydrogen peroxide Test organism 5. Wooden stick or glass rod Reference Documents: Cheesbrough M(2000) District Laboratory Practice in Tropical Countries Part 2 Cambridge University Press, Cambridge, UK. Page 64. Procedure 6.1 Tube technique Pour 2-3ml of the hydrogen peroxide solution into a test tube. Using a sterile wooden stick or a glass rod, remove a good growth of the test organism on the end of the stick and immerse it in the hydrogen peroxide solution. The presence of bubbling indicates a catalase +ve organism. 6.2 Rapid Slide Technique Place a drop of the hydrogen peroxide on a slide Using a wooden stick, pick a growth of the organism to be tested and touch the drop of hydrogen peroxide.
2. 3.
4.
6.0
The presence of bubbling indicates a catalase +ve organism CAUTION: There is risk of contamination of the bench, hands etc., from active bubbling, so conduct the test in an isolated environment.
ANNEX 5 Detecting Indole production 1. Purpose The purpose of this procedure is to assist in the identification of enterobacteria. Escherichia coli, Proteus vulgaris , Proteus rettgeriandMorganellamorganii decompose Tryptophan (a constituent of peptone) to produce indole. Indole is detected by the addition of Kovacs Reagent which contains P-dimethylaminobenzaldehyde, which reacts with Indole to give a red colored compound. Testing for Indole production is important in the identification of enterobacteria. 2. Scope This Procedure applies to the Bacteriology laboratory. 3. Responsibility The bacteriological technician is responsible for the implementation of this procedure. 4. Reference Documents: Cheesbrough M (2000) District Laboratory Practice in Tropical Countries Part 2 Cambridge University Press, Cambridge, UK. pg 67 5. Requirements Tryptone water Incubator at 300C - 370C Kovacs reagent E. coli as the positive control culture Salmonella sp. as the negative control culture 6. Procedure: Inoculate 2.5ml of sterile tryptone water with the test organism. Incubate overnight at 37C. Bacterial controls should also be included: E. coli as positive control (red colour) and Salmonella sp. as the negative control (no colour development). Add approximately 0.2ml of Kovacs Reagent to the Bijou bottle. Shake gently and allow standing for 5 minutes. A red colour developing in the reagent layer indicates the presence of Indole.
ANNEX 6 Identifying Oxidase positive organisms 1. Purpose The purpose of this procedure is to identify cytochrome oxidase-producing organisms that oxidize the phenylenediamine in the oxidase reagent to a deep purple colour. This assists in the identification of Pseudomonas, Neisseria, Vibrio andPasteurellaspecies which produce cytochrome oxidase enzyme. Scope This procedure applies to the Bacteriology Laboratory Responsibility The Laboratory Head is responsible for the implementation of the procedure. Requirements Oxidase reagent (1%Tetramethyl- p- phenylenediaminedihydrochloride) Applicator stick Filter paper Positive and negative control cultures (Positive: Pseudomonas aeruginosa, Negative:E.coli) Reference Documents: Cheesbrough M(2000) District Laboratory Practice in Tropical Countries Part 2 Cambridge University Press, Cambridge, UK, pg 69 Procedure: Place a piece of filter paper in a clean Petri dish and add 2 to 3 drops of oxidase reagent. Using an applicator stick, remove a colony of the test organism and smear it on the oxidase wet area of the filter paper. Do the same for the controls. Look for the development of a blue-purple colour within 10 seconds. 6.1 Reading the Test Blue-purple colour within 10 seconds POSITIVE test, oxidase produced No blue-purple colour within 10 seconds NEGATIVE test, no oxidase produces Positive control Pseudomonas aeruginosa Negative control Escherichia coli
2. 3.
4.
5.
6.
ANNEX 7 Making sterile Urea agar 1. Purpose This procedure ensures that sterile, enriched and selective Urea culture medium is correctly prepared. 1. Scope This procedure applies to the Bacteriology Laboratory. 2. Responsibility The Bacteriological Technician is responsible for the proper implementation of the procedure. 3. Requirements Urea Agar Base (CM53) Distilled Water 100 measuring cylinder 100 ml Conical Flask Weighing boat Autoclave Autoclave tape Weighing balance ( 0.01g) Spatula Water bath at 45oC 1oC Sterile Urea Solution (SR20) Sterile test tubes Sterile 10 ml pipettes Sterile rubber stoppers pH meter 5. Reference Documents: Oxoid Manual 7th Edition 1995 pp 2-219
6.Procedure Weigh and suspend 2.4 g of Urea Agar Base in 95mls of distilled water.

Bring to boil to dissolve completely. Sterilize by autoclaving at 115C for 20 minutes. Cool to 45oC 1oC in water bath. Medium should not remain at 45 oC for more than 3 hrs Aseptically introduce 5 ml of sterile 40% Urea Solution (SR20) to the cooled Urea Agar Base Mix well. Distribute 10 ml amounts of the mixed media into sterile tubes and allow setting in the slope position at room temperature. Label the tubes and store at 2-80 C.
ANNEX 8 Procedure for Coagulase Test 1. Purpose The procedure is used to differentiate Staphylococcus aureusthat produces the enzyme coagulase from other Staphylococcusspp that do not produce coagulase. 2. Scope This procedure applies to the Bacteriology Laboratories. 3. Responsibility The Bacteriological Technician is responsible for implementation of the procedure 4. Requirements Staphylase Test Reagent Kit (Oxoid, UK) Applicator sticks Clean slides 5.Reference Documents: Cheesbrough M (2000) District Laboratory Practice in Tropical Countries Part 2.Cambridge University Press, Cambridge, UK.pp 65 6. Procedure Carry out Gram stain and catalase tests on the colonies to confirm the presence of Gram-positive, catalase-positive cocci. Shake the Test and Control reagentsvigorously to obtain a homogenous suspension. 6.3 Add a drop of the Test Reagent to one end of a clean slide and a drop of the Control Reagent to the other end. 6.4 Using an applicator stick pick 1 to 3 of the test colonies and emulsify the colonies in the drop of the Test Reagent. Repeat the same for the Control Reagent. 6.5 Mix the contents of the slide by rocking. Observe for agglutination while mixing. 7.0 Results Agglutination Staphylococcus aureus. No agglutination No coagulase produced (coagulase negative Staphylococcus species)
6.1
6.2

ANNEX 9 Procedure for making Tripple Sugar Iron 1.Purpose This procedure ensures that sterile Triple Sugar Iron Agar medium is correctly prepared. 2.Scope This procedure applies to the Bacteriology Laboratory. 3.Responsibility The Senior Bacteriological Technician implementation of the procedure.
is
responsible
for
the
proper
4.Reference Documents: Oxoid Manual 7th Edition 1995 pp 2-211 5. Requirements Triple Sugar Iron Agar (CM277) Distilled Water One litre conical flask One litre measuring cyclinder Weighing boat Calibrated thermometer 100C to +1100C Autoclave Autoclave tape Weighing balance 0.01g Water bath at 450 1oC Test tubes Sterile 10 ml pipettes Rubber stoppers Spatula pH meter 6.Procedure Weigh and suspend 65 g of Triple Sugar Iron Agar in 1 litre of distilled water. Bring to boil to dissolve completely.

Mix well and distribute 10ml amounts into test tubes. Sterilize by autoclaving at 115C for 15 minutes. Cool the media to 450Cin a water bath. The media should not remain at 450C for longer than 3 hrs. Allow the medium to set at room temperature in sloped form with a butt about 1 in. deep. Carry out Quality Control. Label the tubes and store at 5 30C.

ANNEX 10 Procedure for making SterileBaird Parker medium 1. Purpose This procedure ensures that sterile Baird Parker medium is correctly prepared for use in the enumeration of Staphylococcus aureus. 2. Scope This covers the Bacteriology Laboratory. 3. Responsibility The Bacteriology Technician is responsible for the proper implementation of the procedure. 4. Requirements Baird-Parker Agar Base- (Oxoid CM0275) Distilled Water Sterile Petri Dishes Conical flask 1 litre Weighing boat Autoclave Autoclave tape Weighing balance (0.01g) Water bath at 45 1o Spatula Egg yolk Tellurite Emulsion (Oxoid SRS4) Sterile 10 ml graduated pipettes 5. Reference Documents: Oxoid Manual. 7th Edition 1995 pp 2-41 Procedure: Weigh and suspend 63g of Baird-Parker Agar Base in one litre of distilled water Bring to the boil to dissolve the medium completely. Sterilize by autoclaving at 121C for 15 minutes. Cool to 45C and aseptically add 50ml of egg yolk Tellurite Emulsion SRS4.

Mix well before pouring into sterile Petri dishes Mark plates with name and date and leave the plates overnight on the bench. Carry out Quality Assurance. Store at 5 3 C

ANNEX 11: Procedure for Performing DNase Test. 1 Purpose The purpose of the procedure is to differentiate S.aureus which produces the enzyme DNAse from other Staphylococci sp.which do not produce DNAse. 2 Scope This test is used in the Bacteriologylaboratories. 3 Responsibility The Laboratory Head is responsible for the correct implementation of the procedure. 4 Requirements Bunsen burner Wire loop Incubator at 37oC 1oC Hydrochloric acid, 1mol/l (1N) 5 Reference Documents: a. Cheesbrough M(2000) District Laboratory Practice in Tropical Countries Part 2 Cambridge University Press, Cambridge, UK. Page 63. 6 Procedure Divide a DNAse plate into the required number of strips by marking the underside of the plate. Using a sterile loop or swab, inoculate the test and control organisms. Make sure each test area is clearly labeled. Incubate the plates at 35-370C overnight. Cover the surface of the plate with 1N hydrochloric acid solution. Tip off the excess acid. Look for clearing around the colonies within 5 minutes of adding the acid. Results: Clearing around the colonies...............DNAse positive strain ( S. aureus NCTC 10788-03) No clearing around the colonies......DNAse negative strain ( S. epidermidis)
Annex 13 Procedure for preparing Phosphatase Solution 1.Purpose


This procedure ensures that Phosphatase Solution is correctly prepared. 1.Scope This covers the preparation of phosphatase solution from phosphatase solution for Phosphatase test 2.Responsibility The Senior Technician is responsible forthe correct preparation of this reagent. 3.Requirements Freshly distilled water Analytical Balance Refrigerator Phosphatase Reagent (4.nitrophenol phosphate disodium salt) Analar grade 100 ml. volumetric flask Spatula Aluminum foil Buffer - NaHCO3Analar grade Na2CO3Analar grade 4.Reference Documents: The Chemical Analysis of Foods by David Pearson Sixth edition. Pg 437438 5.Procedure: Fill half way the volumetric flask with freshly distilled water Weigh 0.15g NaHCO3 powder and transfer to the 100 ml volumetric flask and shake Weigh 0.35 g of Na 2CO3powder and transfer to the same volumetric flask shake again Weigh 0.15 g. of 4-Nitrophenyl-phosphate Disodium salt and add into the flask shake to dissolve. Make the volume to 100mls freshly distilled water. Label the reagent to include (name of reagent, person who prepared it and the date). Store the solution under refrigerated condition, and discard after 2 weeks or when it turns to bright yellow whichever comes first. Shelf-life 2 week

Annex 12 Procedure for the preparation of NaOH N/9 1.Purpose This procedure ensures that NaOH N/9 is correctly prepared for use in the milk Titratable acidity test 2.Scope This direction covers the preparation of N/9 NaOH for estimations of titrateable acidity. 3.Reference Documents: 7.10 7.11 7.12 KBS 05-30 (2001) Specificationfor Raw Milk. KS 05-34 (2001) Specifications for yoghurt. KS 05 941(2001) Specification for lala.
4.Responsibility The Head of the Laboratory is responsible for the implementation of this procedure. 5.Requirements. 10 ml burette 10 ml graduated pipettes 1 lt. volumetric flask 1 lt. conical flask Sodium hydroxide (NaOH) pellets Analar Grade. N/9 Oxalic acid Analytical Grade Analytical Balance Glass funnel Phenolphthalein indicator 50 ml beaker Distilled water 6.Procedure: 6.1 Weigh 4.5 g. NaOHand transfer into the 1litre volumetric flask. 6.2 Add to the flask approx. 200 ml. distilled water and shake to dissolve.

6.3 Make up the volume to 1,000 ml with distilled water. Stopper the flask and invert several times to mix. 6.4 Prepare N/9 oxalic acid by dissolving 7.004g. Oxalic acid in distilled water in a 1,000 ml volumetric flask and making up to volume with distilled water. 6.5 Fill a 10 ml. burette with the N/9 oxalic acid. 6.6 Pipette 10 ml of the NaOH into a beaker and add two drops of Phenolphthalein indicator. This indicator will give a strong purple colour when added to the NaOH. 6.7 Neutralize the NaOH with the N/9 oxalic acid by titrating until the NaOH solution becomes colourless. This should take just about 10 ml. of oxalic acid. Record the volume of oxalic acid used. If the titre is more than 10 ml, then NaOH is of a higher molarity than the oxalic acid and requires dilution.
Calculation of the required dilution to achieve N/9 NaOH Example: If the amount of oxalic acid used was say 11.6 ml, then every 10 ml of NaOH requires 1.16ml distilled water to achieve N/9 NaOH. There is 990 ml. NaOH remaining in the volumetric flask. To bring this to N/9 normality divide 990 by 10 and multiply by 1.16 mleg.: 990 x 1.16 = 114.8 ml 10 Add 114.8 ml distilled water to the volumetric flask and mix well. Take a further 10 ml NaOH into a clean beaker, add two drops of Phenolphthalein indicator and titrate against N/9 oxalic acid. If the concentration is correct it will take exactly 10mls of oxalic acid to neutralize the NaOH. 6.9 Transfer the NaOH solution to a 1 lt. amber glass bottle and label it (name and concentration of solution, date and the name of the person who prepared it) 6.8
END

Training Manual For Food Testing, Microbiology and Physical Chemistry Course UPDATED 33

Hochgeladen von

Dokumentinformationen

Originalbeschreibung:

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Training Manual For Food Testing, Microbiology and Physical Chemistry Course UPDATED 33

Hochgeladen von

Copyright:

Verfügbare Formate

Dr.

Ali Mohamed Ali Iye ( Ali kanu)

VENUE: IGAAD SHEIKH TECHNICAL VETERINARY SCHOOL

1 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

3 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

1.0 GOOD LABORATORY PRACTICE

2.0 LABORATORY SAFETY

3.0 PERSONAL HYGIENE

4 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

4.0 LABORATORY DOS AND DONTS

5 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

b) Bacilli ( rod shaped )

6 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.2 Identification of bacteria

5.2.1 Gram Staining

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.3 Media used in bacteriology

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.4 Media preparation procedure

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.5 Sample preparation and dilutions

10 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.5.1 Procedure for sample preparation and dilution

Dr.Ali Mohamed Ali Iye ( Ali kanu)

12 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.6 Total viable counts (TVC)

13 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

14 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Example Volume applied Dilution 1/100 (10-2) Dilution 1/1000 (10-3)

1 ml 278 and 290 colonies 33 and 28 colonies

16 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Records Bacteriology Laboratory Day Book.

5.7 Coliform counts ( plate method )

Dr.Ali Mohamed Ali Iye ( Ali kanu)

See procedure below.

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Dr.Ali Mohamed Ali Iye ( Ali kanu)

6.5 6.6 6.8

6.9 6.10 6.11

20 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Record Result in the Laboratory Day Book

21 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5.8 Yeast and Moulds counts

Dr.Ali Mohamed Ali Iye ( Ali kanu)

23 Mr. Duncan Ndegwa Mr. Joseph Kimari and

Dr.Ali Mohamed Ali Iye ( Ali kanu)

Enter the results in the Laboratory Day Book showing the

Dr.Ali Mohamed Ali Iye ( Ali kanu)

5. 9 Most Probable Number testing ( MPN) in water