Top 3 Experiments on Medical Microbiology

The following points highlight the top three experiments on medical microbiology. The experiments are: 1. Procedure for Isolation and Identifica­tion of Escherichia Coli 2. Procedure for Isolation and Identifica­tion of Salmonella 3. Procedure for Isolation and Identification of Vibiro Cholarae.

Medical Microbiology: Experiment # 1. Procedure for Isolation and Identifica­tion of Escherichia Coli:

Principle:

Tergitol – 7 agar medium (Sodium heptadecyl sulphate) is a highly selective medium for E. coli and other coliform groups to which the addition of Triphenyl tetrazolium chloride permits the confirmation of E. coli.

Requirements:

1. Tergitol – 7 agar medium [Proteose peptone – 5 g, Yeast extract – 3g, Lactose – 10 g, Agar – 15 g, Tergitol – 7-0.1 ml, Bromothymol blue – 0.025 g, Triphenyle Tetrazolium Chloride – 4 ml, Distilled water – 1 lit. pH. – 7.5]

2. Culture tubes,

3. Needle, Petridishes, slides,

4. Gram Stains,

5. Culture Slants,

6. Microscope,

7. Requirements for respective biochemical tests.

Procedure:

Suspected colonies are picked up separately and then suspended in sterilised distilled water, 1 ml, of diluted sample (10^-1) is poured on a sterilised petridish and then melted tergitol – 7 agar medium (about 20 ml.) is gently added to it. The plates are incubated for 16 – 18 hours at 37°C. Then the appearance of colonies is noted.

The colonies having the following characteris­tics may be considered as E. coli. Circular, non-mucoid and flat colony; a circle of yellow with pinkish tinge appearing at the centre of each colony. Then gram staining is performed to test the gram nature of the colonies.

Finally, several biochemical tests are made lo .identify the presence of E. coli in the suspected cultures.

Observations:

1. Morphology of the Colonies: Circular, non-mucoid, yellowish zone at the centre.

2. Gram Nature: Negative.

3. Results of Biochemical tests.

Medical Microbiology: Experiment # 2. Procedure for Isolation and Identifica­tion of Salmonella:

The pathogenic bacterium Salmonella typhosa and other Salmonella species can easily be isolated by plating the organisms in enrichment medium – i.e., by using selenitic broth or tetrathionate broth.

The colony features on selective media are as follows:

The composition of their enrichment medium is given below:

Furthermore, Salmonella can be identified by a number of biochemical tests.

Requirements:

1. Salmonella culture isolated from nature through enrichment technique in lactose broth.

2. MacConkey Agar medium (Peptone – 17 gms, Proteose peptone – 3 gms, Lactose – 10 gms, Bile salt mix – 1.5 gm., NaCl – 5 gm., Neutral red – 0.03 gm., Crystal violet – 0.001 gm., Agar – 15 gms, Distilled water – 1 lit. pH – 7.)

3. Biochemical test media.

4. Sterilised tubes, petridishes.

5. Needle, incubator use.

Procedure:

Suspected Salmonella culture is plated aseptically on MacConkey Agar and inoculated at 37°C for 48 hours. Salmonella colonies will start appearing on the medium surface.

The following tests are performed:

(a) Presumptive tests using acid/alkaline slants.

(b) Urease test.

(c) Beta – galactosidase test.

(d) V. P. test.

(e) Simmon’s citrate – agar test.

For presumptive tests, the organism is inoculated on T.S.I, slant (TSI composition: Beef extract – 3 gm. yeast extract – 3 gm, Peptone – 2 gm, Sodium chloride – 5 g., Lactose – 10 g.,

Sucrose – 10 g., Glucose – 1 g., Fe(SO₄)2. 7H2O – 0.2 g., Sodium thiosulphate – 0.3 g., Phenol red (0.2% solution) – 12 ml., Agar – 12 g., Distilled water – 1 lit., pH – 7.4). These are incubated for 2 days at 37°C. The alkaline slant turns yellow and acid slant turns red.

For urease test, two 3mm loopfuls of presumptive positive TSI agar pure culture are sub-cultured in urea – broth (Urea – 20 g., Yeast extract – 0.1 g., Potassium di-hydrogen phosphate – 9 g„ Na_:HPO₄ – 9.5 g., Phenol red (0.25%) – 4 ml ; Distilled water – 1 lit., pH – 6.8 ± 0.1) and then incubated for 10 days at 37°C. All cultures that give positive test i.e., purple red colour are discarded.

Salmonella are urease – negative i.e., no change in orange colour of medium takes place.

For β-galactosidase test, use a loopful of TSI culture and inoculate a tube containing 2.5 ml. of sterile ONPG broth.

Buffer solution: NaH₂PO₄ – 6.9 gm. NaOH (0.1N) – 3 ml., water 50 ml.

ONPG solution: O.Nitrophcnyl d-glactopyranoside – 80 mg., water – 15 ml.

Buffer (15 ml.) and ONPG solution (5 ml.) are mixed together to get ONPG broth. Incubate the tubes for overnight. A control tube without inoculum is also maintained. β-galactosidase activity is indicated by the formation of yellow colour due to liberation of free orthonitrophenol. In the presence of Salmonella, the colour of the medium will not change.

Then V-P test (Voges-Proscour test) is performed in the usual way as described earlier. Positive V.P. test is indicated by the development of eosin pink colour. Salmonella gives negative test.

In Simmon’s Citrate agar test, the medium (Sodium citrate – 2 g., Ammonium di-hydrogen Phosphate – 0.2 g., Sodium Chloride – 5.5 g., K₂HPO₄ – 1 g., MgSO₄, 7H₂O – 0.2 g., Bromothymol blue – 0.2 g., Agar – 15 g., Distilled water – 1 lit. pH – 6.9) is inoculated by streaking method with Salmonella culture, and incubated at 37°C for 2 days. Salmonella usually gives positive test shown by colony growth and colour change of medium from green to blue.

Results:

The results of the above stated biochemical tests are given below:

Medical Microbiology: Experiment # 3. Procedure for Isolation and Identification of Vibiro Cholarae:

It is one of the common contaminating pathogenic bacteria. It can be isolated from nature by enrichment technique and then identified by several biochemical tests.

Requirements:

1. Media:

(a) Alkaline peptone water. (Peptone – 10 g., Sodium Chloride – 5 g., Distilled water – 1 lit., pH – 8.4).

(b) Thiosulphate Citrate Bile – salts Sucrose agar medium (TCBS): (Yeast extract – 5 g., Peptone – 10 g., Sucrose – 20 g., Na₂S₂O₅. 5H₂O – 10 g., Sodium citrate – 10 g., NaCl – 10 g., Oxgall – 5 g., Sodium cholate – 3 g., Ferric citrate – 1 g., Bromothymol blue – 0.04 g., Thymol blue – 0.04 g., Agar – 15 g., Distilled water – 1 lit. pH – 8.6).

(c) Bile salt agar medium: (Bact. Peptone – 10 g., Meat Extract – 10 g., NaCl – 5 g., Sodium taurocholate – 5 g., Distilled water – 1 lit., Agar – 15 g., pH – 8.6).

(d) Monsur’s Medium: (a) Bile-salt-gelatin: Tryptone – 1 g., NaCl – 1 g., Nataurocholate – 0.5 g., Na₂CO₃ – 0.1 g., Gelatin – 3 g., Agar – 1.5 g., Water – 100 ml. pH – 8.5. (b) Potassium tellurite solution; Potassium tellurite – 0.5 g., Distilled water – 100 ml., Mix. a and b – 100 ml. of solution a and 1 ml. of solution b.

2. Flask, Needle, Incubator, Culture tubes, racks etc.

3. Reagents for biochemical tests (as stated earlier).

Procedure:

A contamination material is aseptically grown in alkaline peptone broth and shaken thoroughly. Then a loopful of the above broth suspension is transferred to the surface of three different plating media:

(a) Thiosulphate citrate bile salts Sucrose agar (TCBS)

(b) Bile salt agar and

(c) Monsur’s medium and incubated at 37°C for 6 – 8 hours.

In other way fresh peptone broth also is simultaneously inoculated and at an interval of 6 – 8 hours, streaking will be made on the three media as mentioned above to isolate the Vibrio colonies.

The characteristic cultural features of Vibrio colonies on enrichment media are noted. Gram staining is also performed. Then a number of biochemical tests viz. acid production, gas production, oxidase activity etc. are made in the usual way.

Observation:

The cultural features on selective media re tabulated below:

The gram nature is gram negative, non-sporing, curved rod forms. The biochemical tests indicate the following features: