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Chapter Contents
Conventional Plate Count Method
Spiral Plate Method
References
The aerobic plate count (APC) is intended to indicate the level of microorganism in a product. Detailed procedures for determining
the APC of foods have been developed by the Association of Official Analytical Chemists (AOAC) (3) and the American Public
Health Association (APHA) (1). The conventional plate count method for examining frozen, chilled, precooked, or prepared foods,
outlined below, conforms to AOAC Official Methods of Analysis, sec. 966.23, with one procedural change (966.23C). The suitable
colony counting range (10) is 25-250. The automated spiral plate count method for the examination of foods and cosmetics (5),
outlined below, conforms to AOAC Official Methods of Analysis, sec. 977.27. For procedural details of the standard plate count, see
ref. 2.Guidelines for calculating and reporting plate counts have been changed to conform with the anticipated changes in the 16th
edition of Standard Methods for the Examination of Dairy Products (2) and the International Dairy Federation (IDF) procedures (6).
C.
resulting in a low count. Report counts less than 25 or more than 250 colonies as estimated aerobic plate counts (EAPC). Use the
following guide:
1.
2.
3.
4.
D.
Normal plates (25-250). Select spreader-free plate(s). Count all colony forming units (CFU), including those of
pinpoint size, on selected plate(s). Record dilution(s) used and total number of colonies counted.
Plates with more than 250 colonies. When number of CFU per plate exceeds 250, for all dilutions, record the
counts as too numerous to count (TNTC) for all but the plate closest to 250, and count CFU in those portions of plate that are
representative of colony distribution. See ref. 2 for detailed guidelines. Mark calculated APC with EAPC to denote that it was
estimated from counts outside 25-250 per plate range (see D-3).
Spreaders. Spreading colonies are usually of 3 distinct types: 1) a chain of colonies, not too distinctly separated,
that appears to be caused by disintegration of a bacterial clump; 2) one that develops in film of water between agar and bottom of
dish; and 3) one that forms in film of water at edge or on surface of agar. If plates prepared from sample have excessive spreader
growth so that (a) area covered by spreaders, including total area of repressed growth, exceeds 50% of plate area, or (b) area of
repressed growth exceeds 25% of plate area, report plates as spreaders. When it is necessary to count plates containing spreaders
not eliminated by (a) or (b) above, count each of the 3 distinct spreader types as one source. For the first type, if only one chain
exists, count it as a single colony. If one or more chains appear to originate from separate sources, count each source as one
colony. Do not count each individual growth in such chains as a separate colony. Types 2 and 3 usually result in distinct colonies
and are counted as such. Combine the spreader count and the colony count to compute the APC.
Plates with no CFU. When plates from all dilutions have no colonies, report APC as less than 1 times the
corresponding lowest dilution used. Mark calculated APC with asterisk to denote that it was estimated from counts outside the 25250 per plate range. When plate(s) from a sample are known to be contaminated or otherwise unsatisfactory, record the result(s) as
laboratory accident (LA).
Computing and recording counts (see refs 6, 8)
To avoid creating a fictitious impression of precision and accuracy when computing APC, report only the first two significant digits.
Round off to two significant figures only at the time of conversion to SPC. For milk samples, when plates for all dilutions have no
colonies, report APC as less than 25 colonies estimated count. Round by raising the second digit to the next highest number when
the third digit is 6, 7, 8, or 9 and use zeros for each successive digit toward the right from the second digit. Round down when the
third digit is 1, 2, 3, or 4. When the third digit is 5, round up when the second digit is odd and round down when the second digit is
even.
Examples
Calculated Count
APC
12,700
13,000
12,400
12,000
15,500
16,000
14,500
14,000
1.
a.
where:
N = Number of colonies per ml or g of product
C = Sum of all colonies on all plates counted
n1 = Number of plates in first dilution counted
n2 = Number of plates in second dilution counted
d = Dilution from which the first counts were obtained
Example
1:100
1:1000
232, 244
33, 28
= 537/0.022
= 24,409
24,000
b.
When counts of duplicate plates fall within and without the 25-250 colony range, use only those counts
that fall within this range.
2.
All plates with fewer than 25 CFU. When plates from both dilutions yield fewer than 25 CFU each, record actual
plate count but record the count as less than 25 1/d when d is the dilution factor for the dilution from which the first counts were
obtained.
Example
Colonies
3.
1:100
1:1000
EAPC/ml (g)
18
<2,500
<2,500
All plates with more than 250 CFU. When plates from both 2 dilutions yield more than 250 CFU each (but fewer
than 100/cm2), estimate the aerobic counts from the plates (EAPC) nearest 250 and multiply by the dilution.
Example
Colonies
1:100
1:1000
EAPC/ml (g)
TNTC
640
640,000
All plates with spreaders and/or laboratory accident. Report respectively as Spreader (SPR), or Laboratory
Accident (LA).
5.
All plates with more than an average of 100 CFU per sq cm. Estimate the APC as greater than 100 times the
highest dilution plated, times the area of the plate. The examples below have an average count of 110 per sq cm.
Example
Colonies/Dilution
1:100
1:1000
EAPC/ml (g)
TNTC
7,150
>6,500,000 EAPC
TNTC
6,490
>5,900,000 EAPC
(a)
(b)
6.
7.
8.
9.
10.
11.
12.
13.
B.
C.
Preparation of samples.
As described in Chapter 1, select that part of sample with smallest amount of connective tissues or fat globules.
D.
E.
Plating procedure
Check stylus tip angle daily and adjust if necessary. (Use vacuum to hold microscope cover slip against face of stylus tip; if cover
slip plane is parallel at about l mm from surface of platform, tip is properly oriented). Liquids are moved through system by vacuum.
Clean stylus tip by rinsing for 1 s with sodium hypochlorite solution followed by sterile dilution water for 1 s before sample
introduction. This rinse procedure between processing of each sample minimizes cross-contamination. After rinsing, draw sample
into tip of Teflon tubing by vacuum applied to 2-way valve. When tubing and syringe are filled with sample, close valve attached to
syringe. Place agar plate on platform, place stylus tip on agar surface, and start motor. During inoculation, label petri plate lid. After
agar has been inoculated, stylus lifts from agar surface and spiral plater automatically stops. Remove inoculated plate from platform
and cover it. Move stylus back to starting position. Vacuum-rinse system with hypochlorite and water, and then introduce new
sample. Invert plates and promptly place them in incubator for 48 3 h at 35 1C.
F.
Sterility controls
Check sterility of spiral plater for each series of samples by plating sterile dilution water. CAUTION: Prepoured plates should not be
contaminated by a surface colony or be below room temperature (water can well-up from agar). They should not be excessively dry,
as indicated by large wrinkles or glazed appearance. They should not have water droplets on surface of agar or differences greater
than 2 mm in agar depth, and they should not be stored at 0-4.4C for longer than l month. Reduced flow rate through tubing
indicates obstructions or material in system. To clear obstructions, remove valve from syringe, insert hand-held syringe with Luer
fitting containing water, and apply pressure. Use alcohol rinse to remove residual material adhering to walls of system. Dissolve
accumulated residue with chromic acid. Rinse well after cleaning.
G.
1.
2.
Counting grid
Description. Use same counting grid for both 100 and 150 mm petri dishes. A mask is supplied for use with 100
mm dishes. Counting grid is divided into 8 equal wedges; each wedge is divided by 4 arcs labeled l, 2, 3, and 4 from outside grid
edge. Other lines within these arcs are added for ease of counting. A segment is the area between 2 arc lines within a wedge.
Number of areas counted (e.g., 3) means number of segments counted within a wedge. Spiral plater deposits sample on agar plate
in the same way each time. The grid relates colonies on spiral plate to the volume in which they were contained. When colonies are
counted with grid, sample volume becomes greater as counting starts at outside edge of plate and proceeds toward center of plate.
Calibration. The volume of sample represented by various parts of the counting grid is shown in operator's
manual that accompanies spiral plater. Grid area constants have been checked by the manufacturer and are accurate. To verify
these values, prepare 11 bacterial concentrations in range of 106-103 cells/ml by making 1:1 dilutions of bacterial suspension (use a
nonspreader). Plate all Incubate both sets of plates for 48 3 h at 35 1C. Calculate concentrations for each dilution. Count spiral
plates over grid surface, using counting rule of 20 (described in H, below), and record number of colonies counted and grid area
over which they were counted. Each spiral colony count for a particular grid area, divided by aerobic count/ml for corresponding
spirally plated bacterial concentrations, indicates volume deposited on that particular grid area. Use the following formula:
H.
To check total volume dispensed by spiral plater, weigh amount dispensed from stylus tip. Collect in tared 5 ml plastic
beaker and weigh on analytical balance ( 0.2 mg).
I.
J.
K.
L.
References
1.
2.
3.
4.
5.
6.
American Public Health Association. 1984. Compendium of Methods for the Microbiological Examination of Foods, 2nd ed.
APHA, Washington, DC
American Public Health Association. 1993. Standard Methods for the Examination of Dairy Products, 16th ed. APHA,
Washington, DC.
Association of Official Analytical Chemists. 1990. Official Methods of Analysis, 15th ed. AOAC, Arlington, VA.
Donnelly, C.B., J.E. Gilchrist, J.T. Peeler, and J.E. Campbell. 1976. Spiral plate count method for the examination of raw
and pasteurized milk. Appl. Environ. Microbiol. 32:21-27.
Gilchrist, J.E., C.B. Donnelly, J.T. Peeler, and J.E. Campbell. 1977. Collaborative study comparing the spiral plate and
aerobic plate count methods. J. Assoc. Off. Anal. Chem. 60:807-812.
International Dairy Federation. 1987. Milk and Milk Products: Enumeration of MicroorganismsColony Count at 3C.
Provisional IDF Standard 100A. IDF, Brussels, Belgium.
7.
Jarvis, B., V.H. Lach, and J.M. Wood. 1977. Evaluation of the spiral plate maker for the enumeration of microorganisms in
foods. J. Appl. Bacteriol. 43:149-157.
8.
Niemela, S. 1983. Statistical evaluation of Results from Quantitative Microbiological Examinations. Report No. 1, 2nd ed.
Nordic Committee in Food Analysis, Uppsala, Sweden.
9.
Tomasiewicz, D.M., D.K. Hotchkiss, G.W. Reinbold, R.B. Read, Jr., and P.A. Hartman. 1980. The most suitable number of
colonies on plates for counting. J. Food Prot. 43:282-286.
10.
Zipkes, M.R., J.E. Gilchrist, and J.T. Peeler. 1981. Comparison of yeast and mold counts by spiral, pour, and streak plate
methods. J. Assoc. Off. Anal. Chem. 64:1465-1469.
Hypertext Source: Bacteriological Analytical Manual, Edition 8, Revision A, 1998. Chapter 3.
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.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
B.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
(b)
PDA (or MEA)
with chlortetracycline
7 days, 30C
(c)
(d)
Anaerobic agar
MLA
2-4 days, 35C
Enrich MLB dilutions for 7 days, at 30C. Purify growth only if no colonies on MLA.
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.
1.
D.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
E.
1.
2.
3.
4.
5.
6.
7.
F.
G.
1.
2.
3.
4.
5.
H.
Liquids. Decimally dilute 1 ml liquid directly into 9 ml modified letheen broth (MLB) in 20 x 150 mm screw-cap
test tube for the 10-1 dilution.
Solids and powders. Aseptically remove and weigh 1 g sample into 20 x 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.
Cream and oil-based products. Aseptically remove and weigh 1 g sample into 20 x 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.
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 10-1dilution if exactly 1 g of sample was obtained.
Anhydrous materials. Treat as in G-2 or G-3, as appropriate.
Microbiological evaluations
Not all analyses described below will necessarily be performed; however, the aerobic plate count, enrichment culture, and the
fungal count should be performed routinely.
1.
Aerobic plate count (APC). Use spread plate technique to facilitate recognition of different colony types and, if
necessary, for differential count. Also use Baird-Parker (BP) or Vogel-Johnson (VJ) agar if Staphylococcus spp. are suspected.
Prepare and label duplicate sets of petri dishes containing modified letheen agar (MLA) and BP agar for samples of 10-1 to 106
dilutions. Add either 5 or 10 ml of prepared cosmetic preparation (see G, above) to 45 or 90 ml, respectively, of MLB, for 102
dilution.
Dilute samples decimally in MLB (NOTE: save dilutions for enrichment step) to obtain complete dilution series from 10-1 to 10-6.
(Begin with 10-2 if all the 10-1 dilution is used up.) Thoroughly mix dilutions and pipet 0.1 ml of each dilution onto surfaces of solid
media in prelabeled petri dishes. Spread inoculum over entire surface with bent glass rod that was first sterilized by dipping in 95%
ethanol and quickly flamed to remove the ethanol. Let medium absorb inoculum before inverting and incubating plates for 48 h at
30 2C (35C for BP plates). Use new spreader for each dilution (at low dilutions) because some product residue may carry over
and adversely affect the flame-sterilization procedure. For effective inoculum absorption, be sure agar surface is dried (30 min at
35C) when agar is freshly made.
Count all colonies in plates containing 25-250 colonies, and record results per dilution counted. Average the colony counts
obtained, and multiply the average by 10 and then by the appropriate dilution factor (10-1 - 10-6). Report results as APC/g (ml)
sample. If plates do not contain 25-250 colonies, record dilution counted and note number of colonies found.
For BP plates, count well-distributed colonies that are convex, shiny black, either with or without a clear zone surrounding the
colony. (NOTE: Coagulase-positive colonies produce clearing, but coagulase-negative colonies may or may not produce clearing. If
coagulase-negative colonies clear, their irregularity reportedly distinguishes them from coagulase-positive colonies.) Select plates
having more than 250 colonies when those at a greater dilution do not contain colonial types described above. Plates from minimal
dilutions having fewer than 25 colonies may also be used if necessary. From each BP plate demonstrating growth, pick one or more
typical colonies to confirm their coagulase reaction. Transfer colonies to agar slants of any suitable maintenance medium, e.g.,
trypticase (tryptic) soy agar (TSA), brain heart infusion (BHI) agar. Incubate slants until growth is evident.
Calculate number of Staphylococcus aureus organisms present by first determining the fraction of colonies tested that are
coagulase positive. Multiply this fraction by average number ofStaphylococcus colonies counted on the BP plates. Multiply the
number obtained by the appropriate dilution factor and report as number of S. aureus/g (ml) sample.
If no colonies are obtained on MLA or BP media, observe already prepared MLB dilutions while enriching them at 30 2C for 7
days. Examine 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 plates 48 h at 30 2C.
2.
3.
4.
Fungi, yeast, and mold plate count. Transfer 0.1 ml portions of dilution series (H-l, above) to appropriately
labeled duplicate plates of either malt extract agar (MEA) or potato dextrose agar (PDA), both containing 40 ppm chlortetracycline.
Spread inoculum over surface of medium with sterile glass spreader rod. After inoculum is absorbed by medium, invert plates,
incubate at 30 2C, and observe daily for 7 days. Average the counts obtained on duplicate plates, multiply by 10 to allow for the
volume plated (0.1 ml), multiply by the dilution factor, and report as yeast or mold count/g (ml) sample. For fungal enrichments
(optional), dilute 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. The latter two agars should both contain 40 ppm
chlortetracycline.
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, prereduced 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 2C; incubate MLA plates aerobically for 2 days at 35 2C 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 35C 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 A.D. Hitchins, FDA, Washington, DC 20204, for
information about how to proceed.
Screening test for total numbers of microorganisms present in used and unused cosmetics. Solid media,
incubation temperatures and times described in H 1-3 can be applied, as appropriate, to samples prepared as shown in G 1-5 to
screen cosmetics for total counts before performing complete microbiological evaluations as described in H 1-3 above. If sample
contains <10 cfu per ml or g product, a screening test using 1 ml of the 10-1 dilution in the pour plate technique should yield negative
growth results. If sample contains <100 cfu per ml or g product, a screening test using 0.1 ml of the 10-1 dilution in the spread plate
technique should yield a negative growth outcome.
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, Washington, DC 20204, 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). Commercial identification kits, e.g., API, Roche, Vitek, Hewlett-Packard (see Appendix 1), are strongly
recommended.
A.
Identification methods
1.
Gram-positive rods. Report aerobic Gram-positive rods as either sporeforming or nonsporeforming. 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:
a.
b.
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 400X or under oil immersion.
Further characterization of Gram-positive rods is generally unnecessary. Consult refs. 7 and 12 if further characterization of these
organisms is required.
2.
Gram-positive cocci. Streak MLA plate from APC media (MLA or BP), incubate 18-24 h at 35 2C, and test
resultant growth for catalase activity and coagulase production (if catalase-positive).
a.
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.
Coagulase test. Inoculate small amount of growth from maintenance slant into 13 x 100 mm tube
containing 0.2 ml BHI broth. Incubate 18-24 h at 35 2C; then add 0.5 ml reconstituted lyophilized rabbit coagulase plasma (with
EDTA) and mix thoroughly. Incubate at 35 2C 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.
b.
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 2C. If organism blackens bile esculin medium and will grow in presence of 6.5% NaCl, report it as
"Group D enterococcus" (Enterococcusspp.). 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 oxidativefermentative (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. epidermidisif 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.
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 2C. 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 2C. 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
42C, 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.
a.
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 35C for 24 2 h. All slants having growth and an alkaline (red) slant and alkaline (red) butt should be considered as
presumptive positive forPseudomonas 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
1.0 g
Ascorbic acid
0.1 g
Distilled water
100 ml
Cut filter paper (Whatman No. 40) into small strips of about 10 x 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 35C 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.
b.
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 35C for 24 2 h and one slant at 42C for 24 2 h.
(NOTE: Eq agar slants to 42C before inoculation, since other species of Pseudomonas may grow slightly at 42C on
nonpreincubated media, but will not grow on prewarmed slants.) Few Pseudomonas spp. other than P. aeruginosa will grow at
42C.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 withAlcaligenes 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 35C for 24 and 48 h. Citrate utilization is indicated by marked turbidity.
Malonate broth. Inoculate malonate broth. Incubate at 35C 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 35C 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 35C 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 25C (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 25C 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.
TSI
Acid butt, alkaline slant, + gas
Not P. aeruginosa
Not P. aeruginosa
Possibly P. aeruginosa
No growth at 42C
Not P. aeruginosa
Growth at 42C
Probably P. aeruginosa
YE agar
Arginine decarboxylase
Negative
Positive
Probably P. aeruginosa
Koser's citrate
No growth
Growth
--
Possibly P. aeruginosa
Negative
--
Positive
Possibly P. aeruginosa
Negative, no gas
Positive
Possibly P. aeruginosa
Negative
Positive
Possibly P. aeruginosa
Polar flagella
Possibly P. aeruginosa
Not P. aeruginosa
Malonate utilization
Nitrate reduction
Motility
Flagellation
Pseudomonas agar F
No fluorescent pigment
Not P. aeruginosa
P. aeruginosa
Pseudomonas agar P
No pigment
Not P. aeruginosa
P. aeruginosa
C.
D.
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.
E.
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.
15. Morse, L.J., H.L. Williams, F.P. Grenn, Jr., E.E. Eldridge, and J.R. Rotta. 1967. Septicemia due
toKlebsiella pneumoniae originating from a hand-cream dispenser. N. Engl. J. Med. 277:472-473.
16. Smart, R., and D.F. Spooner. 1972. Microbiological spoilage in pharmaceuticals and cosmetics. J. Soc. Cosmet. Chem. 23:721737.
17. Tran, T.T., and A.D. Hitchins. 1994. Microbiological survey of shared-use cosmetic test kits available to the public. J. Ind.
Microbiol. 13:389-391.
18. Tran, T.T., A.D. Hitchins, and S.W. Collier. 1990. Direct contact membrane method for evaluating preservative efficacy in solid
cosmetics. Int. J. Cosmet. Sci. 12:175-183.
19. Weaver, R.E., D.G. Hollis, W.A. Clark, and P. Riley. 1983. Revised Tables for the Identification of Unusual Pathogenic GramNegative Bacteria (E.O. King). Centers for Disease Control, Atlanta, GA.
20. Wilson, L.A., and D.G. Ahearn. 1977. Pseudomonas-induced corneal ulcers associated with contaminated eye mascaras. Am.
J. Ophthalmol. 84:112-119.
21. Wilson, L.A., A.J. Jilian, and D.G. Ahearn. 1975. The survival and growth of microorganisms in mascara during use. Am. J.
Ophthalmol. 79:596-601.
Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 23.
3g
Peptone or gelysate
17 g
Lactose
10 g
1.5 g
NaCl
5g
Neutral red
Crystal violet
0.03 g
0.001 g
Agar
Distilled water
13.5 g
1 liter
Suspend ingredients and heat with agitation to dissolve. Boil 1-2 min. Autoclave 15 min at 121C, cool to 45-50C, and pour 20 ml
portions into sterile 15 x 100 mm petri dishes. Dry at room temperature with lids closed.
DO NOT USE WET PLATES. Final pH, 7.1 0.2.
20.0 g
1.4 g
Potassium sulfate
10.0 g
Agar
13.6 g
Cetrimide
0.3 g
Suspend 45.3 g of ingredients or commercial powder (PseudoselTM, BBL; Becton-Dickinson) in 1 liter of purified water. Add 10 ml
glycerol. Mix well. Heat to boiling, agitating frequently; maintain boiling for 1 min to dissolve powder.
Autoclave at 118C for 15 min.
Final pH, 7.2 0.2. Bacto cetrimide agar base plus glycerol (Difco) is a similar medium.
1g
Polypeptone
10 g
NaCl
75 g
Mannitol
10 g
Phenol red
Agar
Distilled water
0.025 g
15 g
1 liter
Heat with agitation to dissolve agar and boil 1 min. Dispense 20 ml portions into 15 x 100 mm petri dishes. Autoclave 15 min at
121C. Final pH, 7.4 0.2.