COLIFORM/ E.

coli
FDA-Bacteriological Analytical Manual, Chapter 4, 2002
INDOLE 1. Inoculate medium and incubate at 350C for 24  2 hrs.
2. Add 0.2 - 0.3 ml of Kovac’s reagent
SIM MEDIUM
6 ml POSITIVE RESULT: distinct red color/ red ring at the upper layer of
16 x 125 mm c. tubes the medium
1. Inoculate medium and incubate at 350C for 48 2 hrs.
VP
1. Transfer 1 ml into a test tube
MR-VP 2. Add 0.6 ml alpha-naphthol solution.
3. Add 0.2 ml of 40%KOH and shake
MR-VP BROTH 4. Stand for 2 hrs.
10 ml POSITIVE RESULT: eosin pink color
20 x 150 mm c. tubes
MR
1. After the VP test, incubate the MR-VP tube an additional 48 2 hrs
2. Add 5 drops of methyl red solution
POSITIVE RESULT: distinct red color
CITRATE

SIMMON CITRATE 1. Inoculate medium and incubate at 350C for 96hrs.

MEDIUM
3 mL POSITIVE RESULT: a change in color of the medium from green to
4-5cm slants and 2-3cm blue
butts
13 x 100 c. tubes

GAS FROM LACTOSE

LST BROTH 1. Inoculate tube and incubate at 350C for 48 2 hrs.
10 ml
16 x 150 mm c. tubes POSITIVE RESULT: gas production or effervescence after agitation
with durnham tube

RESULTS FOR E. coli

IMViC patterns:  biotype I &  biotype 2
Lactose Fermentation: +

Staphylococcus aureus
FDA-Bacteriological Analytical Manual, Chapter 12, January 2001
 1. Transfer suspected s. aureus colonies to BHI broth, emulsify
thoroughly. (Inoculate TSA slants with loopful of BHI suspension
for possible ancillary tests)
2. Incubate tubes (BHI and TSA) at 350C for 18 to 24  hrs (Retain
TSA slants at room temperature for questionable Coagulase Test
COAGULASE TEST results)
3. Add 0.5 ml of coagulase plasma with EDTA to BHI culture and
BHI BROTH mix.
1 mL 4. Incubate at 350C and examine for clot formation for a period of
13 x 100 c. tubes over 6 hrs.

POSITIVE RESULT: clot formation that stays in place when tube is

tilted or inverted
NOTE: partial clotting (2+, 3+) should be subjected to Ancillary
Tests
Ancillary Tests
1. On a glass slide, drop 3.5% H2O2 solution to suspected s. aureus
colonies.
CATALASE TEST
POSITIVE RESULT: production of gas bubbles
1. Inoculate medium heavily (make sure that inoculum reaches
ANAEROBIC
the bottom of the tube)
UTILIZATION OF
2. Cover surface of agar with sterile paraffin oil (25 mm thick)
GLUCOSE
3. Incubate at 370C for 5 days
OF Medium
POSITIVE RESULT: indicator changes to yellow throughout the
0.5% glucose
tube
ANAEROBIC
1. Repeat the procedure for anaerobic glucose utilization but use
UTILIZATION OF
mannitol instead of glucose as carbohydrate in the medium.
MANNITOL
POSITIVE RESULT: indicator changes to yellow throughout the
OF Medium
tube. S. aureus are usually positive but some strains are negative.
0.5% mannitol
RESULTS FOR Staphylococcus aureus

Catalase activity +
Coagulase production +
Anaerobic utilization of
Glucose +
mannitol +
 Salmonella spp.
FDA-Bacteriological Analytical Manual, Chapter 5, 2007
1. Using a sterile inoculating needle, lightly touch the very center of
the colony and inoculate TSI slant by streaking the slant and
stubbing the butt.
2. Without flaming, inoculate LIA slant by stubbing the butt twice and
streaking the slant.
3. Incubate TSI and LIA slants at 350C for 24  2 hrs with loosely
screwed caps.
TRIPLE SUGAR IRON
Note: Store selective plates at 40C.
TSI AGAR
6 mL
Typical Salmonella reaction
4.5 cm slant and
TSI: red (alkaline) slant and yellow (acid) butt, with or without
2 - 3 cm butt
blackening (H2S production)
16 x 125 mm c. tubes
LIA: purple (alkaline) butt with or without blackening (H2S
production).
Discoloration may occur and must not be confused as an acidic
LYSINE IRON
reaction. Consider only a distinct yellow as acidic reaction.
LI AGAR
Potential Salmonella Isolates:
4.5 mL
1. Cultures that give a purple (alkaline) butt in LIA, regardless of TSI
2.5 cm slant and
reaction.
4 cm butt
2. Cultures that give a yellow (acid) butt in LIA and, a red (alkaline)
13 x 100 mm c. tubes
slant and yellow (acid) butt I TSI.
To be discarded:
1. Cultures that give a yellow (acid) butt in LIA and, a yellow (acid)
slant and butt in TSI.

Note: If TSI cultures fail to give typical reactions, repeat test using
colonies from stored selective media.
1. With a sterile needle, inoculate growth from TSI cultures into urea
UREASE TEST broth.
2. Incubate at 350C for 24  2 hrs.
UREA BROTH 3. Always include an uninoculated tube as control.
3 mL
13 x 100 mm c. tubes POSITIVE RESULT: purple – red color
Salmonella cultures are urease negative.
1. With a sterile needle, inoculate growth from TSI cultures into LD
broth.
LYSINE
2. Replace cap tightly and incubate at 350C for 48  2 hrs but
DECARBOXYLASE
examine at 24 hr intervals.
LYSINE
Typical Salmonella reaction: purple (alkaline) color
DECARBOXYLASE
BROTH
Note: If the medium appears discolored (neither purple nor yellow)
5 mL
add a few drops of 0.2% bromcresol purple dye and re-read the
16 x 125 mm c. tubes
reactions.
 PHENOL RED 1. With a sterile needle, inoculate growth from TSI cultures into PR
DULCITOL BROTH Dulcitol broth.
0.5 % DULCITOL 2. Replace cap loosely and incubate at 350C for 48  2 hrs but
examine at 24 hr intervals.
PR DULCITOL BROTH
2 mL base + 0.5 mL POSITIVE RESULT: gas formation in inner fermentation tube and a
dulcitol solution yellow (acid) pH of the medium. Production of acid (yellow medium)
(2.5g in 100 mL dist. water)
13 x 100 mm c. Tubes only should be interpreted as positive reaction.
with durnham tubes Salmonella cultures are PR dulcitol positive.
1. Transfer 3mm loopful of 24 hr trptone broth culture into malonate
broth.
2. Incubate at 350C for 48  2 hrs but examine at 24 hr intervals.
MALONATE BROTH
3. Always include an uninoculated tube as control.
3 mL
13 x 100 mm c. tubes
POSITIVE RESULT: blue color
Salmonella cultures are malonate negative with the exception of
S. arizonae, majority of which are malonate positive.
RESULTS FOR Salmonella
Test Positive Negative Salmonella spp.
1. Glucose (TSI) Yellow butt Red butt +
2. Lysine Purple butt Yellow butt +
decarboxylase
(LIA)
3. H2S (TSI and LIA) Blackening No blackening +
4. Urease Purple-red color No color change -
5. Lysine Purple color Yellow color +
decarboxylase
broth
6. Phenol red dulcitol Yellow color/gas No gas, no color change +
broth
7. KCN broth Growth No growth -
8. Malonate Blue color No color change -
9. Indole Violet color at surface Yellow color at surface -
10. Polyvalent flagellar Agglutination No agglutination +
test
11. Polyvalent somatic Agglutination No agglutination +
test
12. Phenol red lactose Yellow/gas No gas, no color change -
broth
13. Phenol red sucrose Yellow/gas No gas, no color change -
broth
14. Voges-Proskauer Pink-to-red color No color change -
test
15. Methyl red test Diffuse red color Diffuse yellow color +
16. Simmon's citrate Growth, blue color No growth V
V - variable
Bacillus cereus
FDA-Bacteriological Analytical Manual, Chapter 14, 2001 Edition, Updated Feb. 2012
ANAEROBIC 1. Test anaerobic utilization of Glucose.
 2. Inoculate phenol red glucose broth with loopful of cultures.
UTILIZATION OF 3. Incubate anaerobically for 24hr. at 350C.
GLUCOSE
PHENOL RED POSITIVE RESULT: acid is produced as indicated by color change
GLUCOSE BROTH from red to yello w.
3mL Gas formation on Durham tubes.
13x100mm tubes with
inverted Durham tubes Note: Exposure to CO2 formed in anaerobic jar may slightly reduce
pH of the medium. Take partial color change from red
to orange/yellow as negative.
1. Inoculate nitrate broth and incubate for 24hr. at 35°C.
NITRATE REDUCTION 2. Add two drops of nitrite test reagents A: sulfanilic acid reagent (0.8% in 5M
NITRATE BROTH acetic acid) and C: alphanapthol reagent (0.5% in 5M acetic acid).
5mL
POSITIVE RESULT: Development of orange color after 10min.

1. Inoculate medium and incubate at 350C for 48 2 hrs.

VP
1. Transfer 1 ml into a test tube
MR-VP 2. Add 0.6 ml alpha-naphthol solution.
3. Add 0.2 ml of 40%KOH and shake
MR-VP BROTH 4. Stand for 2 hrs.
10 ml POSITIVE RESULT: eosin pink color
20 x 150 mm c. tubes MR
1. After the VP test, incubate the MR-VP tube an additional 48 2
hrs
2. Add 5 drops of methyl red solution
POSITIVE RESULT: distinct red color
DECOMPOSITION
1. Inoculate entire surface of tyrosine agar slant with 3 mm
OF loopful of culture. Incubate slants 48 h at 35°C.
L-TYROSINE 2. Examine negative slants for obvious signs of growth, and
incubate for a total of 7 days before considering as negative.
TYROSINE AGAR
SLANT POSITIVE RESULT: Clearing of medium near growth.
13x100mm tubes with

GROWTH IN
LYSOZYME 1. Inoculate broth with loopful of cultures incubate for 24hr. at
35°C.
0.001% LYSOZYME IN
2. Incubate negative tubes for additional 24hr. before
2.5mL NUTRIENT
discarding.
BROTH
13x100mm tubes with POSITIVE RESULT: Growth in lysozyme broth

RESULTS FOR B. cereus

Catalase +
Acid from glucose +
Reduction of nitrate +
Tyrosine decomposed +
Lysozyme-resistant +