IDENTIFICATION TESTS FOR PURE CULTURES

1.0 Purpose :
To give a procedure to carry out Identification of pure cultures.
2.0 Scope :
This covers the relevant method to carry out identification of various pure culture organisms.
3.0 Responsibility :
Microbiologist QC
4.0 Procedure :
List of cultures available
4.1.0 Aspergillus niger
ATCC 16404
4.1.1 Bacillus subtilis
ATCC 6633
4.1.2 Bacillus pumilus
NCIB 8982
4.1.3 Candida albicans
ATCC 10231
4.1.4 Escherichia Coli
ATCC 8739
4.1.5 Escherichia Coli M200
NCIB 9270
4.1.6 Micrococcus luteus
ATCC 9341
4.1.7 Pseudomonas aeruginosa
ATCC 9027
4.1.8 Salmonella abony
NCTC 6017
4.1.9 Staphylococcus aureus
ATCC 6538
4.2.0 IDENTIFICATION TESTS:
4.2.1 Aspergillus niger :
4.2.2 Carry out staining with Lactophenol Blue and observe under Microscope with low magnifying power.
-Vegetative hyphae should be septate.
- Characteristic fruiting body (fig-1). Fruiting body has several
Hyphae standing erect to form condiophores which are unseptate.
Each condidophore forms a vesicle which branches to form a bottle shaped sterigmata. These sterigmata produces spores or conidia in chains by the process of budding.
4.3.0 Bacillus pumilus & Bacillus subtilis:
On Gram’s staining both species show Gram Positive with Endospore (fig 2). Difference between the 2 species can be made made out by carrying out the biochemical reaction.

Biochemical Reaction - Bacillus subtilis

Nitrate test - Nitrates reduced to Nitrate
Starch hydrolysis - Starch is hydrolysed
Gelatin liquefaction - Gelatin liquefied
Biochemical Reaction - Bacillus pumilus
Nitrate test - Nitrates not reduced
Starch hydrolysis - Starch not hydrolysed
Gelatin liquefaction - Gelatin not liquefied

4.2.7 Fumigation should be supervised by responsible personnel
To insist the operation in the event of any accident.
4.2.8 Persons coming in contact with formaldehyde solution must Wash their hands and faces thoroughly with soap and water Before eating &/ or drinking.
4.2.9 Shut down the air blowers, slit air conditioners, U.V. lamps, and exhaust fans.
4.3.0 Fumigation
4.3.1 Place the required quantity of fumigation bowls in area.
4.3.2 Place 250ml of formaldehyde solution in previously cleaned
4.3.3 Fumigation bin. Add about 50 gms of potassium permanganate and after conforming the evolution of the gas, the place is to be vacated keeping the door closed.
4.3.4 The door should be locked and carry the sign “DANGER KEEPOUT – FUMIGATION IN PROGRESS”
4.3.5 Fumigate for approximately 24 hours.
4.4.0 De-fumigation
4.4.1 After the stipulated duration , put on the exhaust fan and ensure that no traces of formaldehyde is left.
4.4.2 Use dilute Ammonium solution to absorb the fumes & Clean thoroughly the entire area before manufacturing activities with mild detergents & disinfectants.
4.4.3 After fumigation Settle plates to be exposed to ascertain the Environmental bioload of the area.
4.4.4 The bioload at any given place should not exceed the alert limit.

4.6.0 Frequency:
4.6.1 Once in a week.
4.6.2 Frequency may be increased as required based on bio-loads results.
4.7.0 De-fumigation check:
4.7.1 Take a cotton swab and swab approximately 10 x 10 cms of area near the exhaust grill.
4.7.2 Put the swab into the test tube containing 10mg of chromotropic acid in 5ml of purified water, add 2 to 3 drops of Conc. Sulphuric acid
4.7.3 If violet colour appears it indicates that de-fumigation is not complete.
4.7.4 If violet colour does not persist then de-fumigation is complete.
4.8.0 The log sheets should be maintained for fumigation & de-fumigation in their respective log sheets.