Gram Staining

Catalase
Catalase Production
Most aerobes and facultatives that utilize oxygen produce hydrogen peroxide, which is toxic
to their own enzyme systems. Their survival in the presence of this antimetabolite is possible
because they produce an enzyme called catalase, which converts the hydrogen peroxide to
water and oxygen:

It has been postulated that the death of strict anaerobes in the presence of oxygen may be
due to the suicidal act of H2O2 production in the absence of catalase production. The
presence or absence of catalase production is an important means of differentiation between
certain groups of bacteria.






Oxidase
Oxidase Production
The production of oxidase is one of the most significant tests we have for differentiating
certain groups of bacteria. For example, all the Enterobacteriaceae are oxidase-negative and
most species of Pseudomonas are oxidase-positive. Another important group, the Neisseria,
are oxidase producers.
Two methods are described here for performing this test. The first method utilizes the
entire TSA plate; the second method is less demanding in that only a loopful of organisms
from the plate is used.
Both methods are equally reliable.




Motility







Nitrate
Nitrate Reduction
Many facultative bacteria are able to use the oxygen in nitrate as a hydrogen acceptor in
anaerobic respiration, thus converting nitrate to nitrite. This enzymatic reaction is controlled
by an inducible enzyme called nitratase.

Since the presence of free oxygen prevents nitrate reduction, actively multiplying organisms
will use up the oxygen first and then utilize the nitrate. In culturing some organisms, it is
desirable to use anaerobic
methods to ensure nitrate reduction.
Test Procedure The nitrate broth used in this test consists of beef extract, peptone, and
potassium nitrate. To test for nitrite after incubation, we use two reagents designated as Aand
B.
Reagent A contains sulfanilic acid and reagent B contains dimethyl-alpha-naphthylamine. In
the presence of nitrite, these reagents cause the culture to turn red. Negative results must be
confirmed as negative with zinc dust.
The chemical reaction for this enzymatic reaction
s as follows:






Citrate




Indole




Urea

disk diffusion antibiotic sensitivity testing
is a test which uses antibiotic-impregnated wafers to test whether particular bacteria are
susceptible to specific antibiotics.
A known quantity of bacteria are grown on agar plates in the presence of thin wafers
containing relevant antibiotics. If the bacteria are susceptible to a particular antibiotic, an area
of clearing surrounds the wafer where bacteria are not capable of growing (called a zone of
inhibition).

Procedure
1. Using an asceptic technique, place a sterile swab into the broth culture of a specific
organism and then gently remove the excess liquid by gently pressing or rotating the
swab against the inside of the tube.

2. Using the swab, streak the Mueller-Hinton agar plate to form a bacterial lawn.

3. Allow the plate to dry for approximately 5 minutes.


4. Use an Antibiotic Disc Dispenser to dispense disks containing specific antibiotics
onto the plate.

5. Using a flame-sterilized forceps, gently press each disc to the agar to ensure that the
disc is attached to the agar.

6. Plates should be incubated overnight at an incubation temperature of 37C .