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

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.

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.

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.

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.

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.

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.

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, Andrographispaniculata, 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.

(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

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)

10µl0.1ml

Test sample in 100 ml Lactose broth

Chandra Prakash Singh

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

Growth of red, generally non-mucoid colonies of Gram-negative rods,sometimes surrounded by a reddish zone of precipitation, indicates thepresence of E. coli and material passes the test if no such colonies aredetected.

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)

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

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

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 witha 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; thetest is positive if a purple colour is produced within 5-10 seconds. The material passes the test ifcultures of the type described do not appear.

Chandra Prakash Singh

(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

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-soybeanSabouraud 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 toTransferred to

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

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

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

Thanks for your attention

Analytical Diligence Servicesanalyticaldiligenceservices@gmail.com

singhprakash06@gmail.com Chandra Prakash Singh