MICROBIOLOGY: PRIMARY HEALTH CARE

PUBLIC HEALTH
BACTERIOLOGICAL EXAMINATION OF MILK Pasteurization: Maintaining every particle of the milk at sufficient high temperature for a predetermined period to kill off viable non-sporeforming organisms as well as Mycobacterium tuberculosis and Coxiella burnettii. Holder method: Temperature of 61 63 0 Celsius for 30 minutes. High temperature short time method (HTST): 71 73 0Celsius for 15 seconds. Ultra high temperature (UHT): > 1350C for 1 2 seconds. These methods ensure that milk retains flavour and nutrition while killing pathogens. Pasteurized milk must conform to the milk regulation: 1. Standard plate count must not exceed 50 000 organisms / 1 ml milk. 2. Not more than 10 coliforms / 1 ml milk. 3. Zero E.coli. 4. Phosphatase test must be negative with pasteurized milk. Analysis: SPC (Standard plate count), aerobic plate count, mesophilic plate count: This count provides an estimate of the number of viable micro-organisms in milk according to the medium employed as well as the time and temperature of incubation. Prepare the following dilutions from the milk sample: 10 -1, 10-2, 10-3, 10-4, 10-5. Use PO4 buffer as diluent. Pipette 1 ml of above solutions into sterile petri dishes. Tryptone glucose yeast extract agar is melted, then cooled down to 440Celsius to avoid killing off bacteria in the sample solutions. Pour 10 15 ml molten agar into the petri dishes. Rotate dish 5 X in one direction and 5 X in opposite direction. Allow to solidify. Invert the petri dishes and incubate them at 300Celsius for 3 days (75 hrs). Select a plate which shows between 30 300 colonies. Count the colonies on the plate and multiply the number of colonies by the dilution factor. Interpret result according to milk regulation: < 50 000 / 1 ml milk. Coliform testing: Thoroughly mix samples of milk without frothing. Prepare the 1:10 dilution by adding 5 ml of the product to 45 ml of the sterile diluent. The most probable number (MPN) is determined as follow: Inoculate 3 tubes each containing 10 ml double strength brilliant green (DBG) with 10 ml of the

1:10 dilution of the product. Inoculate 3 tubes each containing 10 ml of single strength brilliant green (SBG) with 1 ml of the 1:10 dilution of the product. Inoculate 3 tubes each containing 10 ml of SBG with 1 ml of the 1:100 dilution. Mix carefully. Incubate the inoculated tubes for 48 hours at 30 0Celsius. Read: Look for gas and turbidity in the durham tube (upside down tube). The number of positive tubes for each dilution is used for the MPN of coliform bacteria per 1 ml of product in accordance with table. Interpretation according to milk regulation: must not exceed 10 coliforms per 1 ml of milk. MOST PROBABLE NUMBER Number of positive tubes 1.0 g or 1.0 ml 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 0.1 g or 0.1 ml 0 0 1 1 2 0 0 0 1 1 2 2 3 0 0 0 1 1 1 2 2 2 2 3 3 3 0 0 0 1 1 1 1 2 2 0.01 g or 0.01 ml 0 1 0 1 0 0 1 2 0 1 0 1 0 0 1 2 0 1 2 0 1 2 3 0 1 2 0 1 2 0 1 2 3 0 1 0.0 0.3 0.3 0.6 0.6 0.4 0.7 1.1 0.7 1.1 1.1 1.5 1.6 0.9 1.4 2.0 1.5 2.0 3.0 2.0 3.0 3.5 4.0 3.0 3.5 4.0 2.5 4.0 6.5 4.5 7.5 11.4 16.0 9.5 15.0 MPN of coliforms / 1 ml or 1 g

3 3 3 3 3 3

2 2 3 3 3 3

2 3 0 1 2 3

20.0 30.0 25.0 45.0 110.0 >110.0

E.coli testing (modified Eijkman test): Mix sample well. Inoculate 3 tubes containing 10 ml of DBG with 1 ml of neat milk. Inoculate 3 tryptone waters with 1 ml of neat milk. Incubate for 48 hrs in a 44o C water bath. Look for the presence of gas in the DBG and for indole production. There should be no E.coli in 1 ml of milk. Phosphatase test (Rudgers): This is an indicator test to see if milk has been pasteurized properly and also to detect the possible addition of raw milk to the pasteurized milk. The test is based on the principle that the alkaline phosphatase enzyme present in raw milk liberates phenolphthalein from a phenolphthalein monophosphate when the tests are conducted at a suitable temperature and pH. Pink color with addition of NaOH indicate presence of phenolphthalein. Microbial phosphatase: Phosphatases produced by micro-organisms in dairy products have been demonstrated and are called microbial phosphatases. They are more heat resistant than the alk.phos. present in milk. Test: 1 ml milk in a clean tube. Add 1 drop phosphatrate alkaline. Control: 1 ml milk in a clean tube. Add 1 drop tartrazine phenolpthalein solution. Mix by inversion. Incubate at 370C for 30 min. After incubation add 1 drop of 2.5M NaOH to both tubes, mix and compare visually. Any shade darker than the control is a positive test. Staphylococcus aureus: Inoculate 0.1 ml neat milk into TPEY agar, spread, incubate at 370C (body temp) for 48 hrs. Look for black colonies with opaque zones around them: Black due to reduction of tellurite to tellurium. Opaque zones due to lipolysis and proteolysis. Count the colonies. Overnight on blood agar coagulation. Milk regulation: zero pathogenic organisms. Salmonella / Shigella: Inoculate 0.1 ml of neat milk onto SS agar. 370C overnight. Look for presence of NLF (non-lactose fermenting) colonies. ID Salmonella or Shigella on SS. Milk regulation: zero organisms. Antimicrobial substances: Qualitative detection of antibiotics and other chemotherapeutic agents in milk and food. Method in the form of an agar diffusion test. Peptone dextrose agar are used and B.stereothermophilus spores are seeded into the agar. Flood a seperate filter paper disc with milk. For each test place a disc on the agar. Use a Pg disc in the center for a positive control.

Incubate in a humidity chamber in a 600C waterbath until it changes color from purple to yellow. Spores utilize the dextrose as they grow and the resulting pH change alters the color. If antimicrobial agents are present the organism will be inhibited around the disc and clear zone forms around them. BACTERIAL EXAMINATION OF WATER Filtration method for the presence of coloiforms: The micro-organisms present in a defined quantity of water are collected on an impervious membrane filter, transferred to a culture medium, incubated and counted. Use a cellulose acetate membrane filter with a pore size of 0.45 m. They should preferably be grid marked in such a manner that bacterial growth is neither inhibited nor stimulated along the grid lines. Method: 1. After sterilization, the filtering apparatus is inserted into the suction jar attached to the vacuum pump. 2. Using sterile forceps, place a sterile filter over the sintered bronze filter bed. 3. Take a sterile rubber ring and place over membrane filter to keep it in place. 4. Carefully place the matched funnel unit over the receptacle and lock it into place. 5. Suitable amount of water to be examined (50 ml or 100 ml) is poured into the funnel. 6. Apply the vacuum; the water filters through the membrane into the flask below. 7. Unlock and remove the filter with sterile forceps and place into m-Endoles agar. 8. Use a new sterile funnel for each specimen. 9. Incubate m-Endoles agar for 22 24 hrs at 350C. Results: Typical coliform colonies have a pink to dark-red color with a metallic surface sheen. Count these colonies and report as coliforms present. Test these colonies for E.coli by putting up a Singers medium. After good growth perform a modified Eijkman test. Must be less than 5 coliforms per 100 ml of water. Modified Eijkman: A tryptone water is inoculated as well as a brilliant green bile broth, and incubated for 48 hrs in a 44oCelsius waterbath. SBG: turbidity and gas. Tryptone water: indole positive. There should be zero E.coli per 100 ml of water. SABS regulation 241 for drinking water standards: Total viable count: < 100 organisms / 1 ml. Coliforms: < 5 / 100 ml. E.coli: absent.

STD SCREENING Urethral and vaginal swabs for Gonococci: Do swabs as soon as possible. Swabs containing transport media will keep the GC alive for longer. All swabs will get the same culture media: BA-agar-CO2 Choc-agar-CO2 NYC-agar-CO2. If you get swabs marked high vaginal / PID / cervical, you will put up additional AN media, eg. naladixic acid agar. Do a gram stain: look for pus cells, epithelial cells, GPC, GNB, yeast or IEGNDC (intracell. gram neg. diplococci). Do a wet prep.: look for Trichomonas vaginalis. Incubate plates overnight and read next day. GC will grow on BA, Choc, NYC (GC colonies will be sticky and round). If there are GNDC present follow culture / sens. method as for GC. Syphilis serology: VDRL (Venereal Disease Research Laboratory) 3 distinct antibodies appear in the serum after a syphilitic infection: 1. Non-specific antibody called reagin. 2. Reacts with a protein component found in the spirochaetal bodies of a non-pathogenic strain of Treponema pallidum. 3. Reacts directly with a pathogenic strain of T.pallidum. The VDRL test detects the presence of syphilitic reagin by means of a reaction between reagin and a standard antigen. The antigen used in this test is composed of cardiolipin and lecithin that have been extracted from beef heart and purified. Cholesterol is added to the alcoholic mix of the cardiolipin and lecithin to increase the antigen's effective surface. Syphilic reagin is capable of producing changes in the dispersion of cardiolipin-lecithin antigen that result in visible flocculation. It is used for screening of serum and CSF samples for syphilis. Rapid reagin tests have been developed eg. RPR detect reagin using a carbon particle antigen; flocculation occurs on rotation on a card if antibody is present results in coagulation of the carbon particles appear as black clumps making macroscopic reading possible. T.pallidum haemagglutination test (TPHA): Passive haemagglutination of erythrocytes is a familiar and very sensitive method for the detection of many antibodies to bacterial and viral antigens. The reagents for this test include formalized tanned sheep red blood cells sensitized with fragments of an ultrasonicated suspension of Nichol's pathogenic strain of T.pallidum. Control unsensitized cells are required, as well as an absorbent and the test diluent. Fluorescent Treponemal antibody test (FTA): This is an indirect immuno-fluorescence technique for detecting antibodies in the serum that react with T.pallidum. Such an antibody may be produced as a result of the presence of commensal treponemes in the body, but most often results from infection with T.pallidum and other pathogenic treponemes. Detects IgG and IgM. High degree of specificity and sensitivity. The patient serum is added to a suspension of dead T.pallidum on a slide. If the patient serum contains antibody against the organism it will combine with the treponemes. To visualise the reaction ie. The presence of human globulin bound on the treponemes an antihuman globulin

antibody tagged with a fluorescent marker (fluorescein isothiocyanate) is added to the carefully washed slide. The mixture is allowed to react and slide washed again. The globulin-coating fluorescence is observed under microscope with UV light. Dark field examination: A dark field microscope is used to read the FTA slides. Use a dry lense X63. The T.pallidum spirochetes will fluoresce and shine, appearing as corkscrew-shaped bodies. The background will be dark. OTHER SEROLOGY TMX: Typhoid fever: Widal agglutination test. Malta fever: Brucella. X Rickettsia: Weil felix. Widal Agglutination test The widal test is used to measure antibodies to O (somatic) and H (flagellar) antigens of the agent of typhoid fever (S.typhii). The antibodies in the patient's serum will agglutinate with the bacterial suspension (antigen). The usefullness of the widal test is greatest when testing on a second specimen of a patient's serum 4 7 days after first specimen. A fourfold rise or greater in O and H antibody titres occurs. O-titre: active infection. High H-titre: past infection or vaccination. 1 / 200: doubtful. 1 / 400: positive. Brucella Agglutination test Used to detect the brucella antibodies in patient serum. The antibodies will agglutinate with brucella antigens. Absence of agglutination does not rule out infection completely because chronic cases give rise to negative results and prozone reactions may occur. 1 / 100: positive. Weil felix Agglutination test: To detect rickettsial antibodies in patient serum. The antigen used is non-specific and represents the O-variant of non-motile strains of Proteus vulgaris OX-19, OX-2, OX-K. These antigens are agglutinated by antibodies developing in blood of patients infected with typhus and certain other rickettsial diseases. South African tick-bite fever: OX-19 pos., OX-2 pos., OX-K neg. 1 / 200: positive. Yersinia Agglutination test: Use specific antigen for detection of antibodies in serum of patients with yersinia infection. RA / RF (Rheumatoid Factor): Latex agglutination test for determination of rheumatoid factors. In an immunochemical agglutination reaction, rheumatoid factors in the patient's serum bind to latex particles coated with human gamma globulin. 20 IU / ml: positive. ASOT (Anti-streptolysin O screen test): Most strains of group A streptococci produce 2 haemolytic factors: streptolysin O and S. Both are capable of haemolysing red blood cells.

If a patient has a gp A infection, streptolysin O will stimulate the development of a specific antibody, anti-streptolysin O, whereas streptolysin S does not stimulate formation of a specific antibody. NB: ASOT may be of diagnostic value in patients having or having had in the recent past, a strep gp A infection. This is of diagnostic importance in Rheumatic fever and Glomerulonephritis. Method: The ASOT test is used for the diagnosis of early rheumatic fever and will dtermine if the antistreptolysin O ab is present in the patient serum. If patient serum is added to the streptolysin O- antigen reagent, the patient antibodies will neutralize the streptolysin O partially or completely, depending on the number of antibodies present in the patient. When red blood cells are added to the free streptolysin O reagent, haemolysis occurs. Consistent quantity of streptolysin reagent is added to progressively diluted amounts of patient serum. If the serum contains sufficient antibody to neutralise the streptolysin O antigen in the reagent, no haemolysis will occur when adding red blood cells to the reagent. At a certain patient dilution where the antigen reagent exceeds patient antibody, the excess streptolysin O cause hemolysis when red blood cells are added. The titre is the reciprocal of the highest dilution of serum which prevents hemolysis of the cells. ANF / ANA (Anti Nuclear Factor antibodies): Antibodies to nucleic acid are screened for most commonly in suspected SLE (Systemic Lupus Erythematosus), and also in chronic active hepatitis. Reaction occurs in 2 steps: 1. The interaction of ANA in patient serum with the cell nuclei. 2. The interaction of fluorescein isothiocyanate (FITC) labelled anti-human gamma globulin with the patient ANA antibodies bound on the cell nuclei. This is a positive assay: the more patient factor bound, the mor pronounced step 2; ie. More fluorescence. Agglutination with neat serum: positive. HIV Sreening: Capillus HIV Latex Agglutination test: Only for detection of antibodies to HIV 1 and 2 in patient serum or plasma. Assay performed on patented capillary agglutination slide which consists of a well area for mixing of latex reagent and sample. At one end of the mixing well is a capillary flow channel which leads to a viewing window. Pos.: agglutination. Neg.: smooth milky white appearrance.

Notes from Cape Pen. University of Technology. More Biomedical Technology Notes at: http://www.scribd.com/people/documents/2135965/folder/83622