Bacteriological Analytical Manual (BAM) Main Page

Authors: Jo Huang, Anthony D. Hitchins (retired), Tony T. Tran (retired), and James E. McCarron (retired)

For additional information, contact Jo Huang or Rebecca Bell

Revision History:

• The original version of this Chapter is available as an archived method.
• July 2017: Revision to Section H-1 and H-2. 
• January 2017: Section H-1 and H-2. Additional 1 ml of 10^-1 dilution is analyzed.
• May 2016: Section H-1. Changed dilution range from 10^-1 - 10^-6 to 10^-1 - 10^-3.
• May 2016: Section H-4. Deleted the entire section: Screening test for total numbers of microorganisms.
• May 2016: Section: Identification of Microbes updated: A.1. Gram-positive rods. Identify Bacillus-like rods if isolated from aerobic plates.
• August 2001: Section: Identification of Microbes. Revised Part D and added reference 2b.
• August 2001: M79 formulation corrected.

The ability of microorganisms to grow and reproduce in cosmetic products has been known for many years. Microorganisms may cause spoilage or chemical changes in cosmetic products and injury to the user (4,5,10,14-16,20,21). Methods for isolation of microorganisms from cosmetic products are direct colony counts and enrichment culturing. Products that are not soluble in water are initially treated to render them miscible before isolation procedures are conducted. Dilution and plating media that partially inactivate preservative systems commonly found in cosmetic products are used. The isolated microorganisms are identified by routine microbiological methods or by commercial identification kits. The scheme for these analyses is summarized in Fig. 1.

A. Equipment and materials

1. Pipets, sterile, 1, 5, and 10 ml, graduated
2. Gauze pads, sterile, 4 × 4 inch
3. Sterile instruments: forceps, scissors, scalpel and blades, spatulas, and microspatulas
4. Test tubes, screw-cap, 13 × 100, 16 × 125, and 20 × 150 mm
5. Dilution bottles, screw-cap
6. Balance, sensitivity of 0.01 g
7. Petri dishes, sterile, plastic, 15 × 100 mm
8. Bent glass rods, sterile
9. Incubators, 30 ± 2°C and 35 ± 2°C
10. Anaerobic atmosphere generating envelopes, indicator strips, and jars (BBL or Oxoid), or anaerobic incubator, 35 ± 2°C, or anaerobic glove box, 35 ± 2°C.
11. Candle jars or CO₂ incubator, 35 ± 2°C.
12. Laminar flow hood with HEPA filter, if available
13. Vitek or equivalent automated computerized identification system

B. Media for enumeration and identification of Gram-positive bacteria and fungi

1. Anaerobe agar (M11)
2. Bile esculin agar (M18)
3. Brain heart infusion (BHI) agar and broth (M24)
4. Malt extract agar (MEA) (M93)
5. Potato dextrose agar (PDA) (M127)
6. Mannitol salt agar (M97)
7. Modified letheen agar (MLA) (M78) and broth (MLB) (M79)
8. Oxidative-fermentative (OF) test medium (M117)
9. Sabouraud's dextrose broth (M133)
10. Blood agar base (M20a)
11. Starch agar (M143)
12. Trypticase (tryptic) soy agar (TSA) (M152) and broth (TSB) (M154)
13. Baird-Parker (BP) agar (M17)
14. Catalase test (R12)
15. Vogel-Johnson (VJ) agar (optional) (M176)
16. Commercial bacterial identification kit (API or equivalent)

Fig. 1: Scheme for Enumeration, Isolation, and Identification of Cosmetic Microbes

Sample preparation.

Dilute prepared samples in MLB.

Spread duplicate 0.l ml samples on..

Count colonies and subculture different colony types on MLA and MacConkey agar (and BP or VJ agars if used in c, above). For fungal isolates, see text.

Determine Gram reaction, cell shape, and catalase production of purified isolates.

Proceed with identification of bacterial isolates as described in text, or use identification kits. 

Fig. l. Abbreviations: MLB, modified letheen broth; MLA, modified letheen agar; PDA, potato dextrose agar; MEA, malt extract agar; BP, Baird-Parker; VJ, Vogel-Johnson.

C. Media for identification of Enterobacteriaceae

1. Andrade's carbohydrate broth and indicator (M13) for testing metabolism of rhamnose, mannitol, sorbitol, arabinose
2. Lysine iron agar (M89)
3. Malonate broth (M92) or phenylalanine malonate broth (Difco)
4. Motility-indole ornithine medium (M99)
5. MR-VP broth (M104)
6. Simmons citrate agar (M138)
7. Triple sugar iron (TSI) agar (M149)
8. Christensen's urea agar (M40)
9. MacConkey agar (M91)
10. Lysine decarboxylase medium for Gram-negative nonfermentative bacteria (M88)
11. Phenylalanine deaminase agar (M123) (see also C-3, above)
12. API 20E, Roche Enterotube, or other equivalent identification kits
    Incubate all biochemical tests using media in B and C, above, at 35-37°C for 18-24 h, except malonate broth (48 h) and MR-VP broth (48 h or longer).

D. Media and reagents for identification of Gram-negative nonfermentative (NF) bacilli

1. Acetamide medium (M2)
2. Clark's flagellar stain (R14)
3. Esculin agar, modified (CDC) (M53)
4. Nutrient gelatin (CDC) (M115)
5. Indole medium (M64) and indole medium (CDC) (M65)
6. King's B medium (M69)
7. Lysine decarboxylase (LDC) medium for Gram-negative NF bacteria (M88)
8. Motility nitrate medium (M101)
9. Nitrate broth, enriched (CDC) (M109)
10. King's OF basal medium (M70) for testing metabolism of sucrose, lactose, fructose, esculin, xylose, glucose (dextrose), mannitol, salicin, sorbitol, and maltose
11. Oxidase test strips
12. Christensen's urea agar (M40)
13. Decarboxylase basal medium (for arginine decarboxylase) (M44)
14. Yeast extract (YE) agar (M181)
15. Pseudomonas agars F (M128 and P (M129) (Difco)
16. Cetrimide agar (Pseudosel^TM, BBL; Difco), or equivalent (M37)
17. Glycerol, sterile (Difco), or equivalent
18. API, NFT, or other equivalent commercial identification system
19. Koser's citrate broth (M72)

E. Other media and reagents

1. Aqueous solution of 70% ethanol and 1% HCl (v/v) or 4% iodine in 70% ethanol solution or 2% glutaraldehyde solution
2. Tween 80 (Polysorb 80)
3. Ethanol, 95% (v/v)
4. Lyophilized rabbit coagulase plasma with EDTA
5. 3% (v/v) Aqueous solution of hydrogen peroxide
6. Gram stain (R32) and endospore stain (R32a)
7. Cooked meat medium (M42)

F. Handling of cosmetic samples for microbiological analysis

Analyze samples as soon as possible after their arrival. If necessary, store samples at room temperature. Do not incubate, refrigerate, or freeze samples before or after analysis. Inspect samples carefully before opening and note any irregularities of sample container. Disinfect surface of sample container with aqueous mixture of 70% ethanol (v/v) and 1% HCl (v/v) or other disinfectant (see E-l) before opening and removing contents. Use laminar flow hood if possible. Dry surface with sterile gauze before opening. Use representative portion of contents for microbial analysis, e.g., 1 g (ml) portion of sample.

For products weighing less than 1 g (ml), analyze entire contents. If only one sample unit is available and multiple analyses are requested (i.e., microbial, toxicological, and chemical), take subsample for microbiological examination before those for other analyses. In this situation, amount of subsample used for microbial analysis will depend on other analyses to be performed. For example, if total sample content is 5 ml, use 1 or 2 ml portion for microbial analyses.

G. Preliminary sample preparation

Amounts of sample and diluent given here can be adjusted according to amount of sample available. If sample has many subsamples, amount of test material can be increased and workload streamlined by compositing. Analysts should use their best judgment as to when and how much material to composite.

1. Liquids. Decimally dilute 1 ml liquid directly into 9 ml modified letheen broth (MLB) in 20 × 150 mm screw-cap test tube for the 10^-1 dilution.
2. Solids and powders. Aseptically remove and weigh 1 g sample into 20 × 150 mm screw-cap test tube containing 1 ml sterile Tween 80. Disperse product in Tween 80 with sterile spatula. Add 8 ml sterile MLB and mix thoroughly. This will be the 10^-1 dilution.
3. Cream and oil-based products. Aseptically remove and weigh 1 g sample into 20 × 150 mm screw-cap tube containing 1 ml sterile Tween 80 plus five to seven 5-mm glass beads (or ten to fifteen 3-mm glass beads). Mix total contents with Vortex mixer. Adjust total volume to 10 ml with sterile MLB (8 ml) for the 10^-1 dilution.
4. Aerosols of powders, soaps, liquids, and other materials. Decontaminate nozzle of spray can as much as possible by swabbing with gauze pad moistened with 70% (v/v) aqueous ethanol. Expel some product to flush out nozzle; then spray appropriate amount into tared dilution bottle, e.g., 1 g of product into 9 ml sterile MLB. Thoroughly mix product and broth, and reweigh. This will be a 10^-1 dilution if exactly 1 g of sample was obtained.
5. Anhydrous materials. Treat as in G-2 or G-3, as appropriate.

H. Microbiological evaluations

1. Aerobic plate count (APC).

Use spread plate technique to facilitate recognition of different colony types. Decimally dilute the cosmetic preparation (see G, above) in MLB to obtain a complete dilution series from 10^-1 to 10^-3. Use a new sterile pipet to transfer 1.0 ml of the current dilution into 9 ml of fresh MLB to make the next decimal dilution.

Mix the dilutions thoroughly and perform all plating in duplicate. Label petri dishes containing modified letheen agar (MLA) accordingly.

For each dilution (10^-1 to 10^-3 dilution), spread 0.1 ml onto MLA. The dilution factors are 100, 1000, and 10,000, respectively.

In addition, plate 1 ml of 10^-1 dilution onto one set of either two (0.5 ml each), or three (0.3, 0.3, 0.4 ml each) MLA plates. Regardless of the number of plates used in plating, the total volume should add up to 1 ml. Do the same for a duplicate set. The dilution factor is 10.

Conduct plating in the same manner for fungi count in H-2, and anaerobic count in H-3 if needed. Save all dilutions for Enrichment step (see below).

Spread the inoculum using a sterile spreader; use a new spreader for each dilution. Let the MLA medium absorb the inoculum before inverting and incubating the plates for 48 h at 30 ± 2°C.
(Tip: To facilitate quick absorption of the inoculum, dry the MLA plates 48 h at 30°C before use.)

Count the colonies from each aerobic plate containing 0.1 ml of the 10^-1 to 10^-3 dilutions and record the numbers. For each set of plates with a total of 1 ml of 10-1 dilution, add the number of colonies and record it and do the same for the duplicate set of plates. Calculate and report aerobic plate counts by following the instructions in BAM Chapter 3.  Aerobic Plate Count, Sections C and D of the Conventional Plate Count Method. Report results as APC/g (ml). If no colonies are obtained on MLA, report the APC as < 10="" cfu/g="">

Enrichment step: Incubate the remaining 10^-1, 10^-2, and 10^-3 dilutions in MLB at 30 ± 2°C for up to 7 days to detect the presence of high-virulence microbial pathogens. Examine the MLB enrichments daily for growth. After 7 days of incubation, or when growth is suspected, subculture all enrichments onto both MLA and MacConkey agar plates. Incubate the plates 48 h at 30 ± 2°C.

2. Fungi, yeast, and mold plant count (YMPC).

Follow plating instructions in H-1 for APC to determine the number of fungi by using either malt extract agar (MEA) or potato dextrose agar (PDA), both containing 40 ppm chlortetracycline.

After the inoculum is absorbed by the medium, incubate the plates at 30 ± 2°C (do not invert and do not stack more than 3 plates high).

(Note: the following plate count instructions are based on BAM Chapter 18, Enumeration of Yeasts and Molds in Food-Dilution Plating Technique.) Count colonies after 5 days of incubation. If there is no growth after 5 days, re-incubate for another 48 h. Do not count colonies before the end of the incubation period because handling of the plates could result in secondary growth from dislodged spores, making final counts invalid. Count colonies on the plates and calculate their numbers as instructed in section H-1, except that the suggested count range is 10-150 colonies. Report as count/g (ml) for yeast and for mold, respectively. If plates from all dilutions have no colonies, report YMPC as < 10="" cfu/g="">

OPTIONAL: For fungal enrichments, dilute the prepared sample decimally in Sabouraud's dextrose broth and incubate as described above for MLB dilutions. If growth occurs, streak on Sabouraud's dextrose agar, MEA, or PDA. Both MEA and PDA agars should contain 40 ppm chlortetracycline.

3. Anaerobic plate count (use only for talcs and powders).

The main purpose of this procedure is to detect the tetanus bacillus (Clostridium tetani), which can occur in these products. Perform as described above for APC, using MLA agar, pre-reduced anaerobe agar, and 5% defibrinated sheep blood agar for plating. Incubate blood agar plates in 5-10% carbon dioxide atmosphere (candle jar or CO₂ incubator), and anaerobe agar plates in anaerobic jars. Incubate both for 48 h before counting. Reincubate for 2 more days if no colonies appear at 48 h. Pre-reduce anaerobic agar plates before inoculation by placing them in an anaerobic atmosphere overnight (12-16 h). Incubate anaerobe agar plates in anaerobic atmosphere (anaerobic jar, incubator, or glove box) for 2 days at 35 ± 2°C; incubate MLA plates aerobically for 2 days at 35 ± 2°C as aerobic control. Strict anaerobes will grow only in the anaerobic jars. It is recommended that a small amount (0.1 ml) of inoculum be used to minimize spreading of growth caused by wetness, and that inoculated plates be placed in an anaerobic atmosphere within minutes after inoculation to minimize exposure to oxygen. Suspected anaerobic organismsmust be subcultured aerobically (under CO₂) and anaerobically to establish their true oxygen relationship. Check for terminally located spores in cooked meat broth incubated at 35°C for 2 days. Use of a differential spore stain to detect spores is mandatory. Other methods may detect nonspore artifacts, which could lead to wasted identification efforts. If an obligate anaerobic sporeformer is isolated, consult Dr. Shashi Sharma, FDA, 5001 Campus Drive, College Park, MD 20740, for information about how to proceed.

Identification of Microbes

Molds and yeasts should be purified and yeasts identified as far as possible using kits, e.g., Vitek yeast card and API yeast assimilation strip. If necessary, send fungal isolates to Valerie H. Tournas, FDA HFS-711, 5001 Campus Drive College Park, MD 20740, for speciation. For bacteria, examine all plates and streak morpho-logically dissimilar colonial types onto MacConkey and MLA media. Prepare Gram stain of all morphologically dissimilar colonial types obtained in pure culture. With methods given here, isolates may be identified to genus level in general; tests for speciation are listed when necessary. Test results should be evaluated using Bergey's Manual (12) or Madden's methods (14). All isolates are required to be identified to the species level.  Commercial identification kits, e.g., API, Roche, Vitek, Hewlett-Packard (see Appendix 1), are strongly recommended to speciate microbes recovered from enumeration plates and enrichment broth.

A. Identification methods

1. Gram-positive rods. 

To enhance sporulation, inoculate starch agar plate with isolate and incubate 48 h at room temperature. Prepare either Gram stain or endospore stain from isolated colony and note position of endospore within vegetative cell (central, terminal, or subterminal), shape of endospore (round or ellipsoidal), and morphology of sporulating cell's sporangium (swollen or not swollen). Test all aerobic sporeforming rods for motility by either of two methods:

Consult refs. 7 and 12 when needed.

1. Cultivation method. Stab-inoculate tube of motility test or motility-indole-ornithine medium. Incubate aerobically 18-24 h at room temperature. Growth from line of stab (indicated by turbidity of medium around stab) constitutes a positive test.
2. Microscopic examination. Inoculate isolated colony into suitable broth. Incubate aerobically 18-24 h at room temperature. Place one drop of broth culture on clean microscope slide and cover with coverslip. Motility is indicated by individual bacterial cells moving in random directions. Observe at either 400× or under oil immersion. 

   Further characterization of Gram-positive rods can be done by performing catalase test then following the testing below:

   1. If catalase is produced and large bacilli (with spores) are observed, use VITEK BCL card, API 50 CHB or equivalent commercial kit to identify the isolate to the species level, possibly Bacillus. When Bacillus cereus group is identified as a group, consult BAM Chapter 14, Bacillus cereus to further distinguish the species in the group. However, only need to identify Bacillus-like rods when the isolates are recovered from aerobic plates, but not from enrichment.
   2. If no catalase is produced, use VITEC CBC card, API 50 CHL or equivalent commercial kit to identify the isolate to the species level, possibly Lactobacillus.
   3. (Rarely) If catalase is produced and short cocco-bacilli are observed, perform motility test. If an isolate is motile at 20-25°C, non-motile at 37 °C, it is possibly Listeria. Use VITEK GP card, API Listeria or equivalent commercial kit to identify the isolate to the species level.

2. Gram-positive cocci.

Streak MLA plate from APC media (MLA or BP), incubate 18-24 h at 35 ± 2°C, and test resultant growth for catalase activity and coagulase production (if catalase-positive).

If no catalase is produced, inoculate bile esculin agar slant, a tube of TSB containing 6.5% NaCl, and a 5% sheep blood agar plate. Incubate 18-24 h at 35 ± 2°C. If organism blackens bile esculin medium and will grow in presence of 6.5% NaCl, report it as "Group D enterococcus" (Enterococcus spp.). If it blackens bile esculin medium, but will not grow in presence of 6.5% NaCl, report it as "Group D Streptococcus, not enterococcus." If it does not blacken bile esculin medium, report it as either alpha, beta, or gamma hemolytic Streptococcus. If 5% sheep blood agar is not available, report it as "Streptococcus, not Group D." Perform additional speciation of streptococci if required, using procedures outlined in ref. 7 or serological kits commercially available for this purpose, e.g., Phadebact (Pharmacia Diagnostics, Piscataway, NJ).

If catalase is produced, inoculate the following media with freshly isolated colony: mannitol salt agar, duplicate tubes of oxidative-fermentative (OF) medium with dextrose (overlay 1 tube with sterile vaspar or mineral oil; leave 1 tube loosely capped with no overlay), and enriched agar slant for use in coagulase test.

Report organism as S. aureus if it is coagulase-positive and/or will ferment mannitol; S. epidermidis if it is fermentative as well as oxidative on OF dextrose, is coagulase-negative, and will not ferment mannitol; or Micrococcus species if it is oxidative only on OF dextrose.

1. Catalase test. Add a drop of 3% H₂O₂ either to isolated colony or to clean microscope slide and place platinum loop carrying some isolate into the drop. Reaction is positive if oxygen gas evolves rapidly (bubble formation). (Nichrome wire loops may give false-positive reactions.) When H₂O₂ is placed directly on a colony the bacteria will be killed. Positive control (Staphylococcus or an enteric bacterium) and negative control (Streptococcus) should be run concurrently to ensure the quality of the H₂O₂ solution.
2. Coagulase test. Inoculate small amount of growth from maintenance slant into 13 × 100 mm tube containing 0.2 ml BHI broth. Incubate 18-24 h at 35 ± 2°C; then add 0.5 ml reconstituted lyophilized rabbit coagulase plasma (with EDTA) and mix thoroughly. Incubate at 35 ± 2°C for 6 h and examine for clotting. Weakly coagulase-producing strains may require overnight incubation for clot formation to be evident. Include known coagulase-positive and known coagulase-negative organism with every set of samples. Consider all strains that yield positive coagulase reaction as S. aureus.

3. Gram-negative rods.

Inoculate TSI agar slant, MacConkey agar plate, cetrimide agar, and MLA plate with all Gram-negative rods. Incubate 18-24 h at 35 ± 2°C. TSI slant/butt reactions of A/A or K/A (A = acidic; K = alkaline) + H₂S indicate an Enterobacteriaceae isolate. K/K, K/NC (NC = no change) or NC/NC reactions indicate nonfermentative (NF) Gram-negative bacilli. If TSI reactions are masked by hydrogen sulfide production, inoculate lactose and glucose carbohydrate broths and incubate 18-24 h at 35 ± 2°C. For an Enterobacteriaceae isolate, perf the following tests and use refs. 3, 6, 11-13 to interpret results. The API 20E or equivalent commercial kit may be used to identify to species level. Media necessary for tests are listed in C, above.

If an organism grows on cetrimide agar or is identified as an NF Gram-negative bacillus, determine fluorescent and nonfluorescent pigment production, aerobic production of acid from either glucose, sucrose, xylose, or mannitol, and the production of nitrogen gas from an inorganic nitrogen source; carry out other necessary tests using media listed in D, above. Acetamide utilization, growth at 42°C, and gelatin liquefaction are important tests for distinguishing the three Pseudomonas species, P. aeruginosa, P. fluorescens, and P. putida. To interpret results of these tests, use refs. 4, 11, 12, 19 or the API nonfermentative kit and data base. Confirm putative P. aeruginosa isolates by the method outlined below.

4. Method for identification of Pseudomonas aeruginosa

Identification of P. aeruginosa is of particular concern because this organism survives in eye-area cosmetics (21) and has been implicated in eye infections (20). It is opportunistically pathogenic to humans (10) and highly resistant to antibacterial agents such as quaternary ammonium compounds, penicillin, and many broad-spectrum antibiotics.

1. Presumptive identification
   
   TSI agar slants. Transfer well-isolated typical colonies from cetrimide agar plates to TSI agar slants. Streak surface and stab butt. Incubate at 35°C for 24 ± 2 h. All slants having growth and an alkaline (red) slant and alkaline (red) butt should be considered as presumptive positive for Pseudomonas spp. and tested for oxidase and other biochemical reactions. Some pseudomonads may produce slight hydrogen sulfide in TSI, but this can be confused with soluble pigments produced by some species.
   
   Oxidase Test
    

   Oxidase test strips (for Pseudomonas spp.)
   Tetramethyl-p-phenylenediamine-dihydrochloride	1.0 g
   Ascorbic acid	0.1 g
   Distilled water	100 ml

   
   Cut filter paper (Whatman No. 40) into small strips of about 10 × 40 mm. Shade in reagent. Drain. Spread strips on paper towels on a tray. Shade with paper towels, because light degrades the reagent; dry in 35°C incubator. (Reagent also degrades at higher temperature.) When dry, store in brown bottle at room temperature. Strips must be protected from light and moisture; they should be white. The strips are stable indefinitely.
   
   Use platinum loop to smear mass of cells on portion of strip. (Nichrome wire gives false-positive reactions.) Read at 10 s, no longer. Positive is indicated by a deep purple color; negative is indicated by the absence of color or when a purple color appears after 10 s. Pseudomonas spp. are oxidase-positive.
    
2. Biochemical tests

   From each positive presumptive TSI agar slant inoculate duplicate YE agar slants, Koser's citrate broth, malonate broth, decarboxylase basal medium containing arginine, motility nitrate agar, nutrient gelatin (CDC), Pseudomonas agar F, and Pseudomonas agar P.

   YE agar slants. Inoculate duplicate YE agar slants. Incubate one slant at 35°C for 24 ± 2 h and one slant at 42°C for 24 ± 2 h. (NOTE: Eq agar slants to 42°C before inoculation, since other species of Pseudomonas may grow slightly at 42°C on nonpreincubated media, but will not grow on prewarmed slants.) Few Pseudomonas spp. other than P. aeruginosa will grow at 42°C. P. aeruginosa produces a fishy odor of trimethylamine on YE agar. Approximately 4% of all cultures routinely encountered fail to produce pigment. Because they are often confused with Alcaligenes spp., Achromobacter spp., or other species, the nonpigmented isolates should be tested further before they are discarded.

   Koser's citrate broth. Inoculate broth and incubate at 35°C for 24 and 48 h. Citrate utilization is indicated by marked turbidity.

   Malonate broth. Inoculate malonate broth. Incubate at 35°C for 24 ± 2 h. A positive test for utilization of malonate as a sole source of carbon is indicated by an indicator change from green to blue (alkaline).

   Nitrate motility agar. To inoculate nitrate motility agar, streak surface and stab butt. Incubate at 35°C for 24 ± 2 h. Add a few drops each of sulfanilic acid and -naphthylamine. A resulting deep pink or red color indicates reduction of nitrate to nitrite. A negative color along with the presence of gas bubbles or cracks in the medium is considered a positive reaction and indicates the reduction of nitrate to nitrite to free nitrogen. NOTE: Always test a control tube incubated under the same conditions.

   Arginine decarboxylase broth. Inoculate decarboxylase basal medium containing arginine. Screw down caps tightly to prevent aeration. Incubate at 35°C for 24 ± 2 h. Examine for growth. A positive reaction for arginine decarboxylation is indicated by no change in color of purple medium. A negative reaction is indicated by an indicator change to yellow (acid).

   Gelatin liquefaction. Inoculate nutrient gelatin tubes and stab butt. Incubate at 25°C (room temperature) for at least 72 h. Chill before examining for liquefaction. Incubate negative tubes 1 week. Negative tubes are normally kept up to 6 weeks before discarding but clearly this is not practical. However, P. aeruginosa usually liquefies gelatin rapidly.

   Pseudomonas agar F and Pseudomonas agar P. Inoculate poured plates of agar F and agar P by streaking. Incubate at 25°C for at least 3 days. Examine agar F with black light (longwave UV). Fluorescent water-soluble pigments (pyoverdines) will diffuse into agar adjacent to streaks containing Pseudomonas spp.

   Break up agar P with glass rod in approximately equal amount of distilled water and shake vigorously until water has removed as much pigment as possible. Decant into separator. In a chemical hood, add 5-10 ml chloroform to water in separator and shake (venting occasionally to prevent internal pressure). The blue pyocyanine will migrate to chloroform. Draw off chloroform layer into test tube. Add about 3 ml distilled water. Add 1 drop 1 N H₂SO₄. Pyocyanine becomes red and migrates to water. Discard solvent in special chloroform-waste bottle.

   Flagella stain. If culture meets all other requirements for P. aeruginosa, including pigments, flagella stain is not needed. Use Clark methods or follow any other suitable method (7), e.g., Leifson or Bailey methods. A rapid wet mount method, using Ryu stain (8) has been reported. P. aeruginosa has a single polar flagellum. The other fluorescent pseudomonads have several flagella.

B. Biochemical results. Examine data in order as shown. Do not skip.

C. Interpretation

Cosmetic products are not expected to be aseptic; however, they must be completely free of high-virulence microbial pathogens, and the total number of aerobic microorganisms per gram must be low. Since there are no widely acceptable standards for numbers, temporary guidelines are used instead. For eye-area products, counts should not be greater than 500 colony forming units (CFU)/g; for non-eye-area products, counts should not be greater than 1000 CFU/g. The presence of pathogens would be particularly important in evaluating as unacceptable a cosmetic with a marginally acceptable count, e.g., 400 CFU/g for an eye-area product. Pathogens or opportunistic pathogens whose incidence would be of particular concern, especially in eye-area cosmetic products, include S. aureus, Streptococcus pyogenes, P. aeruginosa and other species, and Klebsiella pneumoniae. Some microbes normally regarded as nonpathogenic may be opportunistically pathogenic, e.g., in wounds.

D. Cosmetic preservative efficacy

The above guidelines for interpretation of results apply to cosmetic products before the time of use. Cosmetics contain antimicrobial preservatives and thus are expected to withstand a certain amount of abuse by users. Formerly, there were no validated tests for cosmetic preservative efficacy (9), although the test for pharmaceutical preservative efficacy in the U.S. Pharmacopeia (2) or the cosmetic test in the technical guidelines of the Cosmetic, Toiletry, and Fragrance Association (CTFA) (1) were used. Recently, the CTFA test has been AOAC validated (2b) for use with liquid cosmetics. A test for solid cosmetic preservative efficacy has been proposed (18). Cosmetics in reusable test kits, such as those in retail stores, can be microbiologically evaluated semiquantitatively by a sterile swab test (17).

References

1. Anonymous. 1985. Preservation testing of aqueous liquid and semi-liquid eye cosmetics. In: CTFA Technical Guidelines. The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, DC.

2. Anonymous. 1990. Antimicrobial preservatives - effectiveness. In: United States Pharmacopeia, 22nd Revision, p. 1478. U.S. Pharmacopeial Convention, Rockville, MD.

2b. AOAC INTERNATIONAL. 2000. Official Methods of Analysis, 17th ed., Method 998.10. AOAC INTERNATIONAL, Gaithersburg, MD.

3. Brenner, D.J., J.J. Farmer, F.W. Hickman, M.A. Asbury, and A.G. Steigerwalt. 1977. Taxonomic and Nomenclature Changes in Enterobacteriaceae. Centers for Disease Control, Atlanta, GA.

4. De Navarre, M.G. 1941. The Chemistry and Manufacture of Cosmetics. Van Nostrand, New York.

5. Dunningan, A.P. 1968. Microbiological control of cosmetics. Drug Cosmet. Ind. 102:43-45, 152-158.

6. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.

7. Gerhardt, P., R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, and G.R. Phillips. 1981. Manual of Methods for General Bacteriology. American Society for Microbiology, Washington, DC.

8. Heimbrook, M.E., W.L.L. Wang, and G. Campbell. 1989. Staining bacterial flagella easily. J. Clin. Microbiol. 27:2612-2615.

9. Hitchins, A.D. 1993. Cosmetic preservation and safety: FDA Status. J. Assoc. Food Drug Officials 57:42-49.

10. Iglewski, B. 1989. Probing Pseudomonas aeruginosa, an opportunistic pathogen. ASM News 55:303-307.

11. King, E.O. 1964 (revised 1972). The Identification of Unusual Pathogenic Gram-Negative Bacteria. Centers for Disease Control, Atlanta, GA.

12. Krieg, N.R., and J.G. Holt. 1984. Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore.

13. Balows, A., W.J. Hausler, Jr., K.L. Herrmann, H.D. Isenberg, and H.J. Shadomy. 1991. Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, DC.

14. Madden, J.M. 1984. Microbiological methods for cosmetics, pp. 573-603. In: Cosmetic and Drug Preservation: Principles and Practice. J.J. Kabara (ed). Marcel Dekker, New York and Basel.

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Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 23.