PRACTICAL NO-1

PREPARATION OF DIFFERENT CULTURE MEDIA

A growth medium or culture medium is a liquid or gel designed to support the growth
of microorganisms or cells, or small plants like the moss Physcomitrella patens. There are
different types of media for growing different types of cells.
There are two major types of growth media: those used for cell culture, which use specific cell
types derived from plants or animals, and microbiological culture, which are used for growing
microorganisms, such as bacteria or yeast. The most common growth media for microorganisms
are nutrient broths and agar plates; specialized media are sometimes required for microorganism
and cell culture growth. Some organisms, termed fastidious organisms, require specialized
environments due to complex nutritional requirements. Viruses, for example, are obligate
intracellular parasites and require a growth medium containing living cells.

CONSTITUENTS OF BASAL CULTURE MEDIA

1. Water- sources of hydrogen and oxygen.
2. Electrolyte- sodium chloride or other electrolytes
3. Peptone- It is a complex mixture of partially digested proteins prepared from animal or
vegetable proteins by enzymatic action. Commercial preparations of peptone (Bacto-
peptone) contains peptones, proteases, polypeptides, amino acids and inorganic salts
including phosphates, minerals (K, Mg) and accessory growth factores like riboflavin.
4. Meat extract, yeast extract- these contain protein degradation products, carbohydrates,
inorganic salts and certain growth factors.
5. Blood- It enriches media. Usually, 5-10% defibrinated horse or sheep blood is used,
sometimes serum is also required.
6. Agar- it is derived from sea weed (Algae-Geladium species), contains mainly complex
polysaccharides, a small amount of protein like material and sometimes traces of long
chain fatty acids and a variety of impurities including inorganic salts. It is used in 2-3%
concentration as dried fibres as well as powder. It melts at 950C and usually solidifies at
420C. Agar acts merely as a solidifying agent of culture medium and doesnot provide any
nutrition to the bacteria. Moreover, pathogenic bacteria cannot metabolize agar.

TYPES OF MEDIA
1. Chemically defined media: in these, the exact amunt of each ingredient is known. They
are used mainly for experimental purposes but citrate broth is an example of a chemically
defined medium that is used in diagnostic bacteriology.
2. Basic nutritive media: these are capable of sustaining growth of the less fastidious
bacteria. Nutrient agar is an example.
3. Enriched media: agar media, such as blood agar, for the growth of fastidious bacteria.
The media are usually enriched with blood, serum or egg yolk.
4. Enrichment broths: liquid media that are selective for a particular bacterium, such as
selenite broth for the selection of salmonellae.
5. Selective media: these agar media have been made selective for the growth of a particular
bacterium or group of bacteria and are used extensively in diagnostic bacteriology. They
contain inhibitory substances that prevent the growth of unwanted bacterial species.
Many selective media, such as BGA and MacConkey agars.
 6. Indicator media: these are particularly useful in diagnostic bacteriology. They are
designed to give a presumptive identification of bacterial colonies due to the biochemical
reactions in the media. Indicator media often contain ferementable sugars plus a PH
indicator that gives a colour change in the media. MacConkey agar contains the
fermentable sugar lactose and neutral red as the PH indicator. Other indicator media may
be designed to show hydrogen sulphide production (XLD agar) or aesculin hydrolysis
(Edwards medium). Blood agar, although an enriched medium, may also be considered as
an indicator medium as it shows the type of haemolysis of a particular bacterium.
PREPARATION OF CULTURE MEDIA
Requirements for preparation of media in microbiology laboratory
a. Refrigerator for storing media, reagents and species.
b. Small incubator to run at 37± 10C and a maximum-minimum thermometer.
c. Autoclave with pressure gauze and safety valve, which can be set at a temperature of 115
or 1210C.
d. Digital weighing machine that displays minimum of two digits after decimal.
e. Water bath to run at 50-550C and a thermometer to check the temperature.
f. Hot plate and a magnetic stirrer for heating and mixing media or alternatively a tripod
stand and a metal gauze for use with the Bunsen burner. A thick glass rod could be used
for stirring.
g. Sterile plastic petri-dishes of triple vent type or alternatively reusable sterilizable glass
petri-dishes (90 mm) diameter or sterilized test tubes for preparation of liquid media and
stab inoculation media.
Steps for preparing media
a. Dissolve base media in distilled water (w/v) as per manufacturer instruction.
b. Those media that are readily soluble could be autoclaved soon after, for others it is
necessary to dissolve the media in distilled water by alternative heating and stirring. After
complete dissolution solution changes to nearby transparent.
c. Autoclave the solution then after at 1210C and 15 lb atmospheric pressure for 15 min in
autoclave machine. Autoclaving helps to sterilize the solution and completely dissolve
media in distilled water as well.
d. To include antibiotics or blood in the media, cool the autoclaved medium to 50-55°C (a
temp at which flask containing medium is cool enough to be held in hand, yet hot enough
so it remains liquid).
e. Add appropriate amount of a sterile antibiotic solution to achieve desired concentration or
5% (vol/vol) sterile defibrinated blood that has been warmed to room temperature.
Antibiotic solutions are sterilized by filtration through 0.22 μm filter and since blood
cannot be sterilized, it has to be collected aseptically from the animal.
f. After agar has cooled to 45 to 50°C, just above the solidifying point of agar, plates or
tubes may be poured. This is done on the bench, using flame to keep media bottle sterile.
Approx. 50-60 plates can be poured from 1 liter (90 mm sterile petri dishes). If bubbles
form on the surface of the poured plates, a low Bunsen flame should be quickly passed
across the surface of agar before it sets. In case of tube preparation about 3-5 ml of media
must be poured per tube depending upon the type of medium and purpose of test.
g. After the poured plates/tubes are set, they are allowed to dry thoroughly at room
temperature or for few hours in the incubator at 370C. It is better to incubate these plates
overnight at 370C to finally check if the prepared media have any contaminations.
h. For use, the surface of agar must not contain any obvious moisture. The agar could be
used then after or stored at 40C in refrigerator to extend its self-life. Once prepared media
must be used as soon as possible or maximum within 2 weeks of preparation for best
results.
 Practical No. 2

DETERMINATION OF COLONY FORMING UNIT (CFU)

CFU is an estimate of viable bacterial or fungal numbers. Unlike direct microscopic counts
where all cells, dead and living, are counted, CFU estimates viable cells. The appearance of a
visible colony requires significant growth of the initial cells plated - at the time of counting the
colonies it is not possible to determine if the colony arose from one cell or 1,000 cells. Therefore,
the results are given as CFU/mL (colony-forming units per milliliter) for liquids, and CFU/g
(colony-forming units per gram) for solids.

METHODS OF DETERMINING CFU

 The Pour Plate method wherein the sample is suspended in a petri dish using molten
agar cooled to approximately 40-45°C (just above the point of solidification to minimize
heat-induced cell death). After the nutrient agar solidifies the plate is incubated.
 The Spread Plate method wherein the sample (in a small volume) is spread across the
surface of a nutrient agar plate and allowed to dry before incubation for counting.
 The Miles and Misra Methods or drop-plate method wherein a very small aliquot
(usually about 10 microliters) of sample from each dilution in series is dropped onto a
petridish. The drop dish must be read while the colonies are very small to prevent the loss
of CFU as they grow together.
 The Membrane Filter method wherein the sample is filtered through a membrane filter,
then the filter placed on the surface of a nutrient agar plate (bacteria side up). During
incubation nutrients leach up through the filter to support the growing cells. As the
surface area of most filters is less than that of a standard petridish, the linear range of the
plate count will be less.

PROCEDURE OF FINDING CFU

Though there are a variety of variations on this method, the plate count method is the standard
method used in microbiology to estimate CFU. Only plates (or replicate plates from the same
dilution) with 30-300 colonies are counted. Plates with fewer than 30 colonies give statistically
unreliable results, while plates with more than 300 colonies are too crowded to allow all the
bacteria to form distinct colonies. Usually, more than one dilution in a series is plated, just to be
sure that results in a countable range will be obtained. Ignore dilutions giving results outside of
the countable range.

1. SAMPLE DILUTION
Typically ten-fold dilutions are used, and the dilution series is plated in replicates of 2 or 3 over
the chosen range of dilutions.
An advantage to this method is that different microbial species may give rise to colonies that are
clearly different from each other, both microscopically and macroscopically. The colony
morphology can be great use in the identification of the microorganism present.
  The additional 9 ml volumes in each dilution above are of NS or PBS.

2.PLATING VOLUME
0.1 ml volumes is often used when surface-plating, as larger volumes may not be absorbed by the
agar.
3.INCUBATION
Incubate at 37o C for 24-48 hours.
4.COUNT, RECORD, CALCULATE
 Count all colonies. A magnifying colony counter can aid in counting small embedded
colonies.
 Counting colonies is traditionally performed manually using a pencil and a click-counter.
This is generally a straightforward task, but can become very laborious and time
consuming when many plates have to be enumerated. Alternatively semi-automatic
(software) and automatic (hardware + software) solutions can be used.
 When the colonies are too numerous, it is frequent to count CFUs only on a fraction of
the dish. It is finally multiplied by the number of fractions made in the plate to find final
number of colonies in that plate.
 Record the data. Calculate CFU/mL or CFU/g.

CFU/ mL = CFU/plate x dilution factor x 1/aliquot

Suppose a milk sample was diluted 1 to 100 (10 2), 1.0 mL of the dilution was plated and 40
colonies formed. Therefore the count per mL in the milk was:
40 colonies x 102 x 1/1 = 4 x 103 CFU/mL
 Practical No. 3

Common Biochemical Tests for Identification of Bacteria

GRAM STAINING
 1st step in identification of bacteria
 Is a step of differentiating bacteria into 2 large groups: gram +ve and gram –ve
 Based on chemical and physical properties of bacterial cell wall. Primarily it detects the
peptidoglycan present as thick layer in gm+ve bacteria

METHOD
1. Preparing grease free slide
Microscope slides are not always clean enough to use directly from the supplier. Rubbing
with a clean, soft cloth and a flick through the Bunsen flame may be sufficient to remove
a greasy film. If not, a mildly abrasive liquid cleaner can be used followed by rinsing the
slide thoroughly and wiping it dry with a clean cloth.
2. Preparation of smear
Smear could be prepared either from culture made in vitro or directly from field
specimen. It is better to use broth culture for smear preparation. But for the purpose of
immediate analysis a smear should be made directly from field collection as broth culture
is time consuming and is unfruitful in cases that need immediate analysis.
A blunt scalpel and forceps should be kept in a container of 70% ethyl alcohol. The
instruments are flamed and cooled before use. Afterwards they should be placed into a
container of disinfectant. When making a smear from tissue lesions, the specimen is held
firmly with the forceps and the scalpel is used to scrape deep into the material. A small
amount of the scrapings is placed on the cleaned microscope slide. Another clean slide is
used with a scissor action to prepare a thin smear. With liquid or semi-liquid specimens, a
little of the sample is placed on the slide with a sterile swab. The contents of the swab are
smeared over the surface of the slide, with the aim of having thick and thin areas of
specimen present. The smears are allowed to dry thoroughly before proceeding further.
3. Fixing the smears
The reason for fixing the smears include killing the vegetative bacteria, rendering them
permeable to the stain and ensuring that the material is firmly fixed to the slide.
For routine staining the smears are fixed by passing the slide, smear side up, quickly
through the Bunsen flame two or three times, taking care not to overheat the smear. This
can be tested on the back of the hand; the slide should feel warm but not hot enough to
burn.
4. Staining the smear
The fixed smear are placed on a staining rack over a sink. The staining solutions are
flooded over the entire smear and left on the slide for the appropriate time. Between each
staining reagent the smear is washed under a gently running tap water, excess water
tipped off and the next reagent added. Finally the stained smear is washed and air dried.
 Time for staining solution
Crystal violet: 60 sec
Gram’s iodine: 60 sec
 Gram’s decolouriser: 15 sec
Counter-stain (safronin or dilute carbol fuschin): 60 sec

5. Interpretation
Gram-positive bacteria retain the crystal violet-iodine complex and stain purple-blue.
Gram-negative bacteria are decolourised and are stained red by the counter-stain.
Actions of chemicals during Gram staining
 In aqueous medium crystal violet dissociates into
CV CV+ + Cl-
Ions penetrate cell wall of Gram +ve and Gram –ve cell
 I- or I3- form large complexes with CV and forms Crystal violet-Iodine complex within
inner and outer layer of cell wall. Iodine acts as trapping agent that prevents removal of
CV-I complex from cell.
 When decolourizer like acetone or alcohol is added it interacts with lipids of cell wall and
has two possible outcomes. In gram +ve cell dehydration of cell occurs closing the pores
of cell wall as it shrinks during dehydration. Now, CV-I complex gets trapped inside
thick peptidoglycan layer thus no decolourization occurs. However, in gram -ve cell it
loose its outer lipopolysaccharide membrane and inner thin peptidoglycan layer is left
exposed resulting washing out of CV-I complex from inner cell thus the cell decolourize
and is stained with counter stain.
Limitations of Gram Staining
 Small and slender bacteria like Treponema, Chlamydia, Rickettsia are difficult to stain.
 Mycobacterium has extra waxy content in cell wall which is difficult to stain.
 When over decolourized Gm+ve organism looks like Gm-ve and when under
decolourized Gm-ve looks like Gm+ve.
 During cell division thickness of peptidoglycan decreases, so gm+ve bacteria like
Bacillus, Clostridium don’t stain.
 Some cells may be phagocytosed by polymorphs and may lose their stain easily and
appear as mixture of Gm+ve and –ve (Gm-variable).

KOH TEST
Used in differentiating bacteria into Gm+ve and –ve
Method
 A loopful of culture from non-selective media taken and is mixed with equal amount of
3% KOH on a clean slide.
 Lifting of loop is done at intervals to see if gel has formed or not. If thick, viscid stringy
gel with long mucoid strands formed the bacteria is Gm-ve and if no change is visualized
bacteria is Gm+ve.
Principle
 KOH test is used to dissolve cells
 Gm+ve with thick cell wall of peptidoglycan don’t lyse
 Gm-ve with inner porous and thin cell wall lyse, nucleic acid comes out of cell and
bacterial smear becomes a viscous sticky mass.
CATALASE TEST
Detect the presence of catalase in bacteria which protects it from H2O2 inside host.
H2O2 catalase H2O + O2
H2O2 is a potent oxidizing agent that can damage cell.
Methods
a. 1 ml of 3% H2O2 is added on colonies on Nutrient agar. If prompt effervescence occurs
the bacteria is catalase positive.
b. Alternatively place a loopful of bacteria from Nutrient agar in 1 ml of 3% H2O2 taken in a
test tube. If production of gas bubbles is visualized the bacteria is catalase positive.

Principles
Prompt effervescence and production of gas bubbles are due to production of oxygen gas from
H2O2 by activity of catalase enzyme present in bacteria.

Limitations
Since blood contains catalase , cultures on blood containing media are unsuitable for the test.

OXIDASE TEST
Is used to test whether an organism is an aerobe or not.

Method
 Commercial oxidase discs are available for this test
 Keep 1 or 2 drops of distilled water in oxidase disc just to moisture it
 Rub disc with inoculation loop containing bacterial culture
 If the colour of disc change from white to dark purple/blue the test organism is oxidase
positive.
Principle
 Oxidase test checks for the presence of electron transport chain which is final phase of
aerobic respiration. Electron transport chain consists of cytochrome oxidase enzymes
which finally transport electrons to oxygen during respiration.
 In oxidase test, an artificial final e- acceptor is used in place of oxygen. This is a chemical
that changes color to dark blue/purple when it takes electron from last enzyme
cytochrome oxidase in ETC.

OXIDATION FERMENTATION TEST

 Purpose of this test is to determine whether a bacterium attacks sugar by fermentation or
oxidation.
 Medium is semi-solid and usually contains glucose as test sugar and bromothymol as PH
indicator.
 The uninoculated medium (PH 7.1) is bluish green and if the acid is produced by
bacterium, as a result of glucose utilization the medium becomes yellow.
Method
 Recommended dose: 9.4 gm OF media/1000 ml distilled water.
 Divide @ 100 ml in equal parts and autoclave it.
  Mix 10 ml of 100% dextrose solution with 100 ml prepared media solution to make final
volume 110 ml.
 Divide it into different test tube each with 5 ml and incubate overnight.
 Two tubes of OF medium are heated in a beaker of boiling water immediately before use
to drive off any dissolved oxygen. The tubes are cooled rapidly under cold running water.
Both tubes are stab-inoculated with bacterium.
 A layer of sterile paraffin oil is layered on top of one of the tubes (sealed tube) to the
depth of about 1 cm for creating anaerobic condition and the next one is left as such for
creating aerobic condition.
 The inoculated tubes are incubated at 370 C and examined in 24 hours and then daily for
upto 14 days.

Interpretation

Results Open tube (without Sealed tube (with Examples

paraffin) paraffin)
Unreactive Green Green Bordetella spp.
Oxidation Yellow Green Pseudomonas spp.
Fermentation Yellow Yellow Aeromonas spp.

Reactions (O/F) Type of Bacteria Examples

+/- Obligatory aerobic Bordetella spp.
-/+ Obligatory anaerobic Pseudomonas spp.
+/+ Facultative anaerobe or fermentors Aeromonas spp.

Principle
With aerobe/obligatory aerobe
 Metabolize glucose only in open tube.
 Produce acid only in open tube.
 Yellow color seen in open tube.
With fermentor/facultative anaerobe
 Metabolize glucose in both open and closed tube
 Produces acid in both open and closed tube and gives yellow color.
 Practical No. 5

Haemagglutination Test
Haemagglutination test (HA test) is based on the principle of networking attachment of red blood
cells to antigen or pathogens. The presence and identification of pathogen is possible by HA test.
Chicken RBC is used in HA test.

Procedure:

1. Required Materials
o 1% chicken RBC
o Antigen or virus
o Alsever’s solution
o Phosphate buffer saline

2. Preparation of Alsever’s solution

 D-glucose: 2.05 gm
 Sodium citrate: 0.8 gm
 Sodium chloride: 0.42 gm
 Citric acid: 0.055 gm
 Distilled water: Up to 1000 ml.
Mix all chemicals and autoclave it under 15 lb pressure for 15 minutes.

3. Preparation of Phosphate buffer saline

o Sodium Choride: 0.8 gm
o Potassium chloride: 0.02 gm
o Di-sodium hydrogen phosphate: 0.115 gm
o Potassium di- hydrogen phosphate 0.02 gm
Mix all chemicals and autoclave it under 15 lb pressure for 15 minutes.

4. Preparation of chicken RBC

 Hold chicken suitably exposing sites of wing vein and sterilize the site of blood
collection.
 Take 2 ml blood from wing vein into syringe containing equal volume of
Alsever’s solution.
 Mix blood with phosphate buffer solution in centrifuge tube.
 Centrifuge the blood mixture at 500 rpm for 5 minutes.
 Remove the supernatant solution and again mix with phosphate buffer solution.
 Repeat until the color of supernatant becomes clear.
 Lastly, make 1% chicken RBC taking 1 part washed RBC and 99 parts phosphate
buffer solution.