Drigalsky Microbiology

Glass spatula (Drigalsky)

3. Methods

1. Weigh 10 g of the product under aseptic conditions and add the sample to 90 mL of TAT

or Leethen broth to obtain a dilution of 10-1 (see Note 1).

2. Make dilutions of 12-2 and 10-3 in tubes with 9 mL of the same diluter.

Starting from the dilutions, perform the following counts.

1. Count of mesophilic aerobic microorganism (standard plate-count method).

a. Incubate at 35°C for 48 h. Carry out the reading.

b. Calculate the colony-forming units (CFU)/mL.

c. To duplicate sets of Petri plates, pipet 1-mL aliquots from the 10-1 dilution and add tripticase soy agar with lecithin and polysorbate 80.

d. Agitate gently and appropriately.

e. Add additional layer of agar.

g. Calculate and report the result per gram or milliliter of analyzed cosmetic product.

2. Test for total coliforms MPN.

a. Inoculate three replicate tubes of brilliant green bile (BGB) broth per dilution with 1 mL of the previously prepared 1:10, 1:100, and 1:1000 dilutions.

b. Incubate tubes for 24 and 48±2 h at 35±0.5°C.

c. Observe all tubes for gas production either in the inverted vial (Durham) or by effervescence produced. Read tubes for gas production after 24 h. Reincubate negative tubes for an additional 24 h.

d. Report the results as the MPN of total coliforms per gram or milliliter of sample.

3. Count of molds and yeasts.

a. Pipet 1-mL aliquots from 10-1 to 10-5 dilutions. Promptly put into Petri plates 1015 mL of oxytetracycline gentamicin yeast extract glucose (OGY) agar, melted and tempered to 45°C.

b. Mix and leave to solidify, adding an additional layer of agar (4-5 mL).

d. Count all colonies on plates containing 30-300 colonies; compute the number of yeasts and molds per gram or milliliter of product. Report as CFU per gram or milliliter of sample.

4. Test of absence/presence of Staphylococcus aureus (coagulase positive).

a. Starting from the TAT broth, that was incubated for 4-6 h, inoculate 0.25 mL of a 10-1 dilution on each one of two dishes of Vogel and Johnson agar.

b. Spread the inoculate using a Drigalsky spatula.

d. If there is growth of suspicious colonies, confirm by coagulase test or thermostable endonuclease.

5. Test of absence/presence of Pseudomonas aeruginosa.

a. Repeat the previous procedure, sowing in Cetrimide.

b. Observe the growth and typical characteristics and confirm with an oxidase test (positive).

c. Report the results as a test of abscence or presence.

d. Starting from the TAT broth at a dilution of 10:1 that was incubated for 4-6 h, as a revivification procedure, inoculate 0.25 mL on the surface of each of four Petri dishes with Cetrimide or Pseudosel agar.

e. Carefully spread the inoculate with a Drigalsky spatula.

g. Confirm characteristics compatible with P. aeruginosa with an oxidase test.

4. Notes

1. Letheen broth: Dissolve 0.7 g lecithin and 5.0 g polysorbate 80 or Tween-80 in 400 mL of hot H2O and boil until clear. Add 600 mL solution of 5.0 g beef extact, 10.0 g peptone, and 5.0 g NaCl in H2O, and boil for 10 min. Adjust pH to 7.0 ± 0.2 (with 1 NNaOH or 1 N HCl). Filter through coarse paper, transfer 10-mL portions to test tubes (16 x 150 mm), and autoclave (20 min at 121°C).

References

1. American Public Health Association (1984) Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. (Speck, M. L., ed.). Compiled by the APHA Technical Commitee on Microbiological Methods for Foods. Washington, DC, American Public Health Association.

2. American Public Health Association (1978) Standard Methods for the Examination of Dairy Products, 14th ed. (Marth, E. H., ed.). Washington, DC, American Public Health Association.

3. Code of Federal Regulations (1983) Title 21, Food and Drugs. Par 129, Processing and Bottling of Bottled Drinking Water. Washington, DC, US Govt. Printing Office.

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