1. Total viable aerobic count
Membrane filtration method
Serial dilution method
Test for specific microorganisms
Enterobacteriaceae and certain other
Gram-negative bacteria.
Escherichia coli
Salmonella spp.
Pseudomonas aeruginosa
Staphylococcus aureus
Planning of Microbiological
study for determination of
microbiological load.
Analytical Diligence Services Chandra Prakash Singh

2. Washing & Sterilization of glassware
All glassware was dried in hot air oven and wrapped with brown paper before
sterilization in hot air oven at 180°C for 45 min. All freshly prepared culture
medium was sterilized in autoclave at 121°C for 15min after plugging with non-
adsorbing cotton and wrapped with brown paper. All sterile culture medium and
preparation of agar plate was done in aseptic laminar hood.
Cleaning of glassware was done in three steps; in first step, for removing organic
matter form glass, chromic acid was used and in second step detergent solutions
was used. Prolonged rinsing with water and finally rinsed with purified water
before was adopted at last step.
METHODOLOGY – contd.

3. Washed, dried, wrapped conical flask
keeped for sterilization in hot air oven
Sterilized petridish keeped for
medium transfer in laminar hood
Freshly prepared culture medium
keeped for sterilization in autoclave
METHODOLOGY – contd.

4. 1. Baird-Parker agar
2. Brilliant green agar
3. Buffered sodium chloride-peptone solution pH 7.0
4. Casein-soybean digest agar
5. Cetrimide agar
6. Deoxycholate citrate agar
7. Enterobacteriaceae enrichment broth-Mossel
8. Lactose broth
9. MacConkey agar
10. MacConkey broth
11. Sabouraud glucose agar with antibiotics
12. Soybean-casein digest medium
13. Tetrathionate bile brilliant green broth
14. Violet-red bile agar with glucose and lactose
15. Xylose, lysine, deoxycholate agar
Culture medium for
use in microbiological
study
METHODOLOGY – contd.

5. 1. Microorganism stains and culture medium use in validating the tests for
specific microorganism
Microorganism Stain Number Medium
Escherichia coli MTCC No. – 1687
(Equivalent to ATCC8739)
Lactose broth
Pseudomonas aeruginosa MTCC No. – 1688
(Equivalent to ATCC9027)
Soybean-casein digest
medium
Salmonella enterica ser.
Abony – (Salmonella
typhimurium)
MTCC No. – 3858
(Equivalent to NCTC6017)
Lactose broth
Staphylococcus aureus
subsp.aureus
MTCC No. – 737
(Equivalent to NCTC7447,
ATCC6538P)
Soybean-casein digest
medium
Microorganism stains and culture medium for use in microbiological study
METHODOLOGY – contd.

6. 2. Microorganism stains and culture medium used in study of effectiveness of the
culture medium and validity of counting method
Microorganism Stain Number Medium
Staphylococcus aureus
subsp.aureus
MTCC No. – 737
(Equivalent to NCTC7447,
ATCC6538P)
Soybean-casein digest
medium
Bacillus subtilis MTCC No.- 441
(Equivalent to ATCC6633)
Soybean-casein digest
medium
Escherichia coli MTCC No. – 1687
(Equivalent to ATCC8739)
Soybean-casein digest
medium
Candida albicans MTCC No.- 183
(Equivalent to ATCC2091)
Soybean-casein digest
medium
ATCC = American Type Culture Collection
MTCC = Microbial type culture collection & gene bank
NCTC = National Collection of Type Cultures
METHODOLOGY – contd.

7. Sample ID-A 0 day sample
Sample ID-B 30 days stored sample in metalized film bags
Sample ID-C 30 days stored sample in polythene bags
Sample ID-D 30 days stored sample in woven polypropylene mesh bags
Sample ID-E 30 days stored sample in jute bags
Sample ID-F 120 days stored sample in metalized film bags
Sample ID-G 120 days stored sample in polythene bags
Sample ID-H 120 days stored sample in woven polypropylene mesh bags
Sample ID-I 120 days stored sample in jute bags
Sample ID-J Presence of the material being examined were used for
validating the tests
Sample ID-K Absence of the material being examined were used for
validating the tests
Sample ID
METHODOLOGY – contd.

8. Pretreatment of material being examined for test of specific microorganisms &
total viable aerobic count
Buffered sodium chloride-peptone solution
suspension was used for Pseudomonas aeruginosa,
Staphylococcus aureus, total viable aerobic count
determination.
10g of material
Moderately fine powder (sieve no 355/180) of Withania somnifera, Andrographis
paniculata, Terminalia arjuna, Tinospora cordifolia and Plantago ovata
10g of material
Lactose broth Buffered sodium chloride-peptone
Dilute up to 100ml with the same medium
A suitable surfactant (a solution of polysorbate 80 R containing 0.1 mg per ml) was
added and pH of the suspension was adjusted to about 7.
Incubated at 35°C±2°C for a length of time sufficient for revivification of the bacteria but
not sufficient for multiplication of the organisms (usually 5 hours).
Lactose broth suspension was used for
Enterobacteriaceae & certain other Gram-
negative bacteria, Escherichia coli, Salmonella
spp.

9. (Incubated at 35°C±2°C for 24 hours)
KJIHGFEDCBA
KJIHGFEDCBA
KJIHGFEDCBA
Detection of Enterobacteriaceae and certain other Gram-negative bacteria
(Incubated at 35°C±2°C for 5 hours)
1ml in each 100ml of Enterobacteriaceae enrichment broth-Mossel
(Mixed and incubated at 35°C±2°C for 24 hours)
Subculture was prepared on a plate with violet-red bile agar with glucose and lactose.
material passes the test if no growth of colonies of Gram-negative bacteria is detected
on the plate.
Test sample
in 100 ml
Lactose
broth

10. Quantitative evaluation Enterobacteriaceae and certain other Gram-negative
bacteria
KJIHGFEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
(Incubate at 35°C±2°C for 5 hours)
1ml
In each 100ml of Enterobacteriaceae enrichment broth-Mossel
(Mixed and incubated at 35°C±2°C for 24 hours)
Subculture was prepared on a plate with violet-red bile agar with glucose and lactose.
(Incubated at 35°C±2°C for 24 hours)
10µl
0.1ml
Test sample
in 100 ml
Lactose
broth
Chandra Prakash Singh

11. The growth of well-developed colonies, generally red or reddish in colour, of
Gram-negative bacteria constitutes a positive result. Note the smallest quantity of
material that gives a positive result. Determine the probable number of bacteria
using table.
Result for each quantity or volume Probable number of bacteria per g
of material1.0 ml 0.1 ml 10 µl
+ + + More than 102
+ + - Less than 102 but more than 10
+ - - Less than 10 but more than 1
- - - Less than 1
Quantitative evaluation Enterobacteriaceae and certain other Gram-negative
bacteria
Chandra Prakash Singh

12. Growth of red, generally non-mucoid colonies of Gram-negative rods,
sometimes surrounded by a reddish zone of precipitation, indicates the
presence of E. coli and material passes the test if no such colonies are
detected.
KJIHGFEDCBA
KJIHGFEDCBA
KJIHGFEDCBA
Examination of Escherichia coli
1ml in each 100ml of MacConkey broth
Mixed and incubated at 44°C±2°C for 24 hours.
Subculture was prepared on a plate with MacConkey agar
(Incubate at 35°C±2°C for 5 hours)
Test sample
in 100 ml
Lactose
broth
(Incubated at 44°C±2°C for 24 hours)

13. KJIHGFEDCBA
KJIHGFEDCBA
10ml in each 100ml of tetrathionate bile brilliant green broth
Mixed and incubated at 42°C±2°C for 24 hours
Subculture was prepared on a plate with two set of the following three agar media
(Incubate at 35°C±2°C for 5 hours)
Test sample
in 100 ml
Lactose
broth
Primary test of Salmonella spp.
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
KJIHG
FEDCBA
Deoxycholate citrate agar Xylose, lysine deoxycholate agar Brilliant green agar
Incubated at 35°C±2°C for 36 hours

14. Description of Salmonella colonies appearing on different
culture medium
Medium Description of colony
Deoxycholate citrate agar Well developed, colorless.
Xylose, lysine
deoxycholate agar
Well developed, red, with or without black
centers.
Brilliant green agar Small, transparent and colourless or opaque, pink
or white (Frequently surrounded by a pink to red
zone)
Primary test of Salmonella spp.
Chandra Prakash Singh

15. Estimation of Pseudomonas aeruginosa
(Incubated at 35°C±2°C for 24 hours)
KJIHGFEDCBA
KJIHGFEDCBA
KJIHGFEDCBA
(Incubated at 35°C±2°C for 5 hours)
1ml in each 100ml of Soybean-casein digest medium
(Mixed and incubated at 35°C±2°C for 24 hours)
Subculture was prepared on a plate with Cetrimide agar
If growth of colonies of Gram-negative rods occurs, usually with
a greenish fluorescence, Oxidase test was applied.
Test sample in 100
ml Buffered sodium
chloride-peptone
solution, pH 7.0
Oxidase test - Place 2 or 3 drops of a freshly prepared 0.01 g/ml solution of N,N,N',N'-tetramethyl-
p- phenylenediamine dihydrochloride on filter-paper and apply a smear of the suspected colony; the
test is positive if a purple colour is produced within 5-10 seconds. The material passes the test if
cultures of the type described do not appear.
Chandra Prakash Singh

16. (Incubated at 35°C±2°C for 24 hours)
KJIHGFEDCBA
KJIHGFEDCBA
KJIHGFEDCBA
(Incubated at 35°C±2°C for 5 hours)
1ml in each 100ml of Soybean-casein digest medium
(Mixed and incubated at 35°C±2°C for 24 hours)
Subculture was prepared on a plate with Baird-Parker agar
The material passes the test if no growth of microorganisms is detected.
Black colonies of Gram-positive cocci often surrounded by clear zones indicate
the presence of Staphylococcus aureus.
Test sample in 100
ml Buffered
sodium chloride-
peptone solution,
pH 7.0
Estimation of Staphylococcus aureus

17. Total viable aerobic count by membrane filtration method
Diluted upto ten times
10ml diluted enrichment culture was transferred and washed with filtering three successive
quantities of 100ml of a buffered sodium chloride-peptone solution, pH 7.0.
Cellulose nitrate membrane filters (0.45μm)
Casein-soybean
Sabouraud glucose
agar with antibiotics
Test sample in 100 ml
Buffered sodium
chloride-peptone
solution, pH 7.0
(Incubated at 35°C±2°C for 5 hours)
(For the enumeration of bacteria) (For the enumeration of fungi)
Transferred to
Transferred to

18. Incubated the
plates for 5 days
at 32°C±2°C
Incubated the
plates for 5 days
at 22°C±2°C
The number of colonies
formed was counted.
Number of bacteria per g of the
material tested was calculated.
The number of colonies
formed was counted.
Number of fungi per g of the
material tested was calculated.
Total viable aerobic count per g of the material tested =
Number of bacteria per g + Number of fungi per g

19. Diluted upto ten times and used for further dilution
Test sample in 100 ml
Buffered sodium
chloride-peptone
solution, pH 7.0
(Incubated at 35°C±2°C for 5 hours)
Total viable aerobic count by serial dilution method
1ml of diluents1ml of 1:10 dilution 1ml of 1:100 dilution 1ml of 1:1000 dilution
No microbial growth should appear in the last three tubes and determine the most
probable number of microorganisms per g of the material using table.
+ 9ml of soybean-casein digest medium was added.
The tubes were incubated at 35°C±2°C for 5 days.
1 2 3 4 5 6 7 8 9 10 11 12

20. Number of with microbial growth Most probable no. of
microorganisms per ml0.1 ml per tube 0.01 ml per tube 0.1 ml per tube
3 3 3 1100
3 3 2 1100
3 3 1 500
3 3 0 200
3 2 3 290
3 2 2 210
3 2 1 150
3 2 0 90
3 1 3 160
3 1 2 120
3 1 1 70
3 1 0 40
3 0 3 95
3 0 2 60
3 0 1 40
3 0 0 23
Table for determination of most probable number of microorganisms
per g material
Total viable aerobic count by serial dilution method

21. Thanks for your attention
Analytical Diligence Services
analyticaldiligenceservices@gmail.com
singhprakash06@gmail.com Chandra Prakash Singh