1. Summary of Biochemical Tests in Microbiology
“Better living through chemistry”

2. Catalase Test Summary Tests for presence of catalase. Background
Catalase is an enzyme that breaks down hydrogen peroxide into water and oxygen gas. Evolution of oxygen gas bubbles is positive.
All + except EF –
Methods
Loop of bacteria added to a drop of Hydrogen Peroxide H2O2

3. Blood Agar Test Summary Tests for presence of hemolysin. Background
Hemolysin is an enzyme that destroys red blood cells.
Methods
Bacteria streak and loop stabs to blood agar.

4. 3 Types of Blood Agar Results
Summary
Alpha – Partial hemolysis
Beta – Complete hemolysis
Gamma – No hemolysis
Background
Alpha hemolysis partially oxidizes hemoglobin changing it to green.
All B+ except SE and EF
Beta hemolysis lyses RBC completely making a clear zone
Gamma hemolysis – no activity

5. Mannitol Salts Agar (MSA) Test
Summary
Selective for gram +
Differential for fermentation of mannitol sugar.
Background
Gram + bacteria tolerate sodium chloride (NaCL) salt.
SA + EF + SE - and others –
Methods
Streak surface of MSA Agar. Watch for change from pink to yellow as a +

6. McConkeys Test Summary Tests for fermentation of lactose. Background
Selects for gram - , Crystal violet inhibits gram +
Lactose and neutral red produce a dark pink differentiating lactose +
EC, CLO, KP are +
Methods
Loop of bacteria streaked on Mac agar

7. EMB Agar Test Summary Tests for fermentation of lactose. Background
Selects for gram - , Eosin and methylene blue inhibit gram +
Lactose and dyes produce a dark pink differentiating lactose + in CLO and a green sheen in EC
EC, CLO, KP are +
Methods
Loop of bacteria streaked on EMB agar

8. Nitrate Reduction Test
Summary
Tests for nitrate reductase and nitrite reductase.
Background
Potassium Nitrate KNO3 as substrate. Nitrate  Nitrite  N2
Methods
. Add Nitrate A & B and cherry red means + for nitrate reductase
All +
Follow up with Zinc = grey = + for nitrite reductase
PA +

9. Triple Sugar Iron (TSI) Test
Summary
Differentiates gram – enterics by fermentation patterns.
Background
Lactose and glucose are substrates. Phenol red is the indicator. pH of 6.8 or lower turns yellow.
Methods
Stab and streak TSI slant.
Red slant & yellow but = glucose fermentation. ST SF PA
Yellow slant and yellow butt = lactose and glucose fermentation. SA SE EF BS EC CLO KP PV(false)
Red slant & red butt = no fermentation.
Black butt = Hydrogen sulfide production

10. Urease Test
Summary
Differentiates organisms using urease to break down urea into ammonia
Background
Substrate is urea. pH indicator is phenol red (pH 6= yellow, 6.8 topaz, 8 = fuchsia)
Methods
Loop inoculation of urea broth.
SA SE KP PV are +

11. IMViC Indole Test Summary and Background
Differentiates enteric organisms such as EC and PV which are able to metabolize the amino acid tryptophan into indole and pyruvic acid. This test also detects motility of bacteria and H2S2
production.
Methods
Needle inoculation of Sufide-Indole-Motility (SIM) media. Substrate is tryptophan. Add Kovac’s indole reagent. A red color is + for indole production.
Black = + for H2S2
Motility is observed as turbidity radiating out from the inoculation line.

12. IMViC Methyl Red Summary and Background
Differentiates enteric organisms such as EC from CLO using the timing of glucose fermentation products. Both produce acids but CLO further converts to non acidic ethanol and then elevates the pH.
Methods
Loop inoculation of MR VP broth. Add methyl red indicator.
Red = + for acid conditions. SA SE BS EC KP PV ST SF
Yellow is – indicating more basic conditions. EF CLO PA

13. Citrate Test
Summary
Differentiates organisms using citrate as a carbon source.
Background
Organisms using citrate must also have nitrogen found in the medium as ammonium dihydrogen phosphate. Citrate fermentation produces ammonia causing the indicator (bromothymol blue) to turn deep blue.
Methods
Loop inoculation of citrate slant.
Prussian blue = + for citrate fermentation and alkaline conditions.
Green or yellow = - (low pH)