BACTERIAL GROWTH

Growth is dependent on:

1. Availability of nutrients

2. The external environment

3. Growth rate of the specific species

Growth curve

a-lag phase

b-log or exponential phase

c-stationary phase

d-phase of decline or death

The lag phase is a period of no growth since the organisms are still adapting to their
new environment. The log phase is the steady rate of growth, and this is the time
where antibiotics, especially those targeting cell wall synthesis, are most effective.
It is during this phase that the bacteria need the most nutrients: carbon, nitrogen,
sulfur, phosphate andminerals.

In the stationary phase, all the nutrients have been used, or the toxins
haveacumulated, hence :

Growh may stop, though viability may persist; viability is no possible, thus death
occurs; formation of spores.

In the death phase, cell starvation or toxin sensitivity is detrimental to the bacteria.

SURVIVAL IN OXYGEN

This is also a way of classifying bacteria, since all the bacteria produce superoxide
ion (O2-) in the presence of oxygen. 3 enzymes are need to detoxify the radical:

1. Superoxide dismutase, which converts superoxide ion to hydrogen peroxide

2. Catalase

3. Peroxidase

Catalase and peroxidase convert Hydrogen peroxide to water and oxygen.

PRODUCTION OF ENERGY

Bacteria use a source of carbon for energy. 2 mechanisms may be used:

1. Fermentation is the anaerobic degradation of glucose to obtain ATP; used by

facultative and obligate anaerobes. The end products of fermentation are
useful in aiding identification. Obligate anaerobes generally lack catalase.

2. Respiration requires Electron transport chain so that it may oxidize organic

fuels and synthesis ATP. This produces more energy, compared to
fermentation. Obligate aerobes respire only and use O2 as its terminal
electron receptor.

Some bacteria may require low oxygen tension such as Camphylobacter, and are
termed microaerophilic.

Other anaerobes may tolerate O2 for short periods, and are called aerotolerant
anaerobes, such as Actinomyces.
 GROWTH OF BACTERIA IN OXYGEN

(+)

OBLIGATE AEROBES OBLIGATE

ANAEROBES

FACULTATIVE

SOURCE OF ENERGY

OXYGEN REMOVED

FERMENTS FERMENTS

NO GROWTH (+)GROWTH RESPIRE

OBLIGATE FACULTATIVE CLOSTRIDIUM

BACTEROIDES

AEROBE

(Mycobacteria) SOURCE OF ENERGY

FERMENTS FERMENTS/RESPIRE

STREP E.COLI

CLASSIFICATION BASED ON MOTILITY

Bacteria may also be classified based on their motility. Most pathogenic cocci are
NONMOTILE such as Salmonella, E.coli, Proteus, Vibrio and Pseudomonas. The
hanging drop is the simplest test to determine motility.

GRAM’S STAIN

This is intended for the morphologic study of the bacteria, dividing them into gram
(+) and Gram (-). The following is the widely used procedure:

1. Air dry specimen, and fix the slide.

2. Immerse in crystal violet for 1 min.

3. Wash with iodine for 1 min.

4. Decolorize with alcohol, then wash with water.

5. Immerse in safranin for 1 min.

6. Air dry, then view under oil immersion.

Investigation may then proceed based on morphology, biochemical characteristics

and antibiotic sensitivity.

Note the 2 algorithms used for gram’s stain.

CATALASE TEST is the most useful test to differentiate between Staph and Strep.
Here, hydrogen peroxide is added to a culture sample, and the bacteria’s enzyme
catalse will break it down to water and oxygen, which is visualized as
bubbles(positive result).

COAGULASE TEST determines the presence of coagulase in the bacteria. Coagulase

serves as the defense mechanism since it clots the plasma around the bacteria,
walling it off from the environment.In the lab, this is reproduced by using 0.5ml
rabbit serum, incubated at 37 C, for 1-4 hours. A positive result is the formation of a
clot.

NOVOBIOCIN is an antibiotic which inhibits DNA gyrase, preventing the supercoiling

of DNA.A positive result is the formation of a clear alo around the disc, also known
as the zone of inhibition.

BLOOD AGAR is used for further evaluation of Streptococci. Streptococci species

may have the enzyme Streptolysin, which maycause varying degrees of hemolysis.

Partial (alpha)hemolysis refers to the incomplete breakdown of heme in the blood

agar, thus the agar is colored green.
Complete (beta) hemolysis refers to the complete lysis of the blod cells.

Gamma hemolysis refers to the absence of hemolysis.

OPTOCHIN TEST uses ethyl hdrocupriene, and a positive result is the zone of
inhibition around the disc, which should be more than 14mm.

BILE TEST refers to the solubility of the organism in bile; this is the positive result.

QUELLUNG TEST is based on the reaction of an added antibody to the specimen;

this willreact to the polysaccharide antigen in the specimen’s capsule. A positive
result is the capsule’s swelling.

BACITRACIN is added to specimens which have complete or beta hemolysis on blood

agar. It is an antibiotic produced by Bacillus subtilis var tracy. A positive result is the
zone of inhibition.

GRAM (-) cocci specimens are plated in a special agar, either chocolate agar if it
came from a sterile source, such as the CSF, or using Thayer Martin agar, if the
source was unsterile, such as the cervix. TM agar is chocolate agar with
Vancomycin, Nystatin and Colstin added to prevent the growth of normal vaginal
flora. Once grown in either agar, the biochemical tests can be performed to
determine which is a glucose or glucose+maltose fermenter.

HAEMOPHILUS will grow on chocolate agar; or on agar which has been cross-
streaked with Staphylococcus, since the latter will release factoe V and X. This is
termed satellitism.

Bordetella will grow on Bordet Gengou agar,and then determined further by

serologic methods.

GRAM (-) rods are plated in Eoson Methylene Blue agar, which inhibits the growth of
gram (+) organism. Once grown, the specimen is determined whether it possesses
the ability to ferment lactose.

LACTOSE FERMENTERS like Klebsiella,E.coli and Enterobacter willshow as dark

centered, clear borderd colonies. Non lactose fermenters will show up as colorless
colonies.

OXIDASE TEST detects a component of the cytochrome oxidase complex. A positive

result will be a purple color; absence of color will be a negative result.
COMMON CULTURE MEDIA

H.influenzae – chocolate agar

N. gonorrheae- Thayer martinagar

B. pertussis- Bordet gengou agar

Lactose fementes-EMB agar

M.tuberculosis-Lowenstein Jensen agar

Fungi-Sabouraud’s agar.

IMPORTANT STAINS

ZIEHL NEELSEN STAIN – for acid fast bacteria, such as Mycobacterium; once stained,
it resists decoloration with alcohol since the dye adheres to the mycolic acid in the
bacteria’s cell wall.

INDIA INK STAIN- negative stain since it stains the background black, showing the
bacteria’s capsule; specific for Crytococcus neoformans.

GIEMSA STAIN-for Borellia, Plasmodium, Chlamydia; the stain atachs to the adenine-
thymin bonding sites on the DNA; hence it is ideal for:

1. Staining chromosomes

2. Differential stain for human cells and bacterial cells

3. Malarial/spirochetes/protozoans

4. Peripheral bllod smears

5. Bonne marrow specimens