OBJECTIVES

1. To perform test toward the unknown organisms into the media and biochemical tests.
2. To investigate the results obtained from media and biochemical tests in order to rule out
the unknown organisms given.
3. To find the reliable drugs that can be used to kill the organisms.

RESULTS
MORPHOLOGY ON CULTURE MEDIA

Test Results
Organism Q Organism R Organism S
Haemolysis Alpha Gamma Gamma

Elevation Raised Convex Flat

Density Opaque Opaque Opaque
Margin Irregular Entire Irregular
Consistency Mucoid Mucoid Mucoid
Size Large Medium Very large
Odor Fishy Fishy Fishy
Colour Cream White Yellow
Gram stain Gram negative bacilli Gram positive bacilli Gram positive
coccobacilli
Oxidase - - -
Catalase + - +
BIOCHEMICAL TEST
Tests Results
Organism Q Organism R Organism S
TSI K/K with no gas K/no change with no gas K/A with gas and H2S
production

Motility Motile Non-motile Non-Motile

Indole - - -

Methyl red - - -

Voges - - -
Proskauer
Citrate Positive Positive Negative

PD - - -

OF-glucose Oxidase Oxidase Ferment

OF-sucrose Negative Ferment Fermant

Urea - - -
MAC  Growth, lactose  Growth, lactose No Growth
fermenter (pink fermenter (pink
colonies) colonies)

TCBS  Growth with green No Growth  Growth with green colour

colour colony colony
 Medium  Medium unchanged
unchanged

EMB  Growth, pink  Growth, (colourless No Growth

colour colony colonies)
Medium unchanged  Medium unchanged
 ANTIBIOTIC SENSITIVITY TESTING

Antibiotics Organism Q Organism R Organism S

1. CIP5 Susceptible (3cm) Susceptible (3cm) Susceptible (3cm)

2. CN10 Susceptible (2cm) Susceptible (1.5cm) Susceptible (1.5cm)

3. E15 Resistance (0cm) Susceptible (1.6cm) Susceptible (1.5cm)
4. AMP10 Resistance (0cm) Resistance (0cm) Susceptible (1.5cm)
5. SXT25 Resistance (0cm) Resistance (0cm) Susceptible (2.5cm)

DISSCUSSION

1. What are the different characteristics of non-Enterobacteriaciae versus Enterobacteriaciae

group? (8 marks)
N NON - ENTEROBACTERIACIAE
O ENTEROBACTERIACIAE
1 Not part of normal flora Usually part of normal flora
2 Obligate aerobes Facultative anaerobes
3 Mostly oxidase positive Mostly oxidase negative
4 Do not use carbohydrates as a source Use carbohydrate as aa source of energy
of energy

2. Describe the mode of action of antibiotics against bacteria. (6 marks)

I. Cell Wall Synthesis Inhibitors:
Some antibiotics like beta-lactams, cephalosporins and glycopeptides inhibits the ability
of microorganism to synthesize the cell the wall which mainly contains murein and
peptidoglycan.
II. Protein Synthesis Inhibitors:
 Some antibiotics like Streptomycine, Erythromycine, tetracyclines, chloramphenicol,
lincomycin, kanamycin disulphate salts, aminoglycosides and macrolides interfere in the
formation of protein synthesis of microbes
III. Cell Membrane Inhibitors:
Antibiotics like polymyxins interrupt the integrity and structure of cell membranes, hence
killing them. These types of antibiotics are generally effective on gram –ve bacteria as
they have a definite cell membrane
IV. Effect on Nucleic Acids: 
The whole existence and life of any living system depends on DNA and RNA present in
every living cell. Some antibiotics like etopocide, rifampcin, quinolones and rifamycins
bonded with those proteins which are essential for the processing of these nucleic acids.
Hence these antibiotics block the synthesis of nucleic acid and therefore affect the growth
of the living cells.
V. Competitive Inhibitors: 
Some antibiotics competitively inhibit the important metabolic pathways happen inside
the bacterial cell. These antibiotics resemble a microbial substrate and compete with that
substrate for the limited microbial enzyme. The antibiotic ties up the enzyme and blocks
a step in metabolism. They are also called as anti-metabolites or growth factor analogs.
For example; Sulfonamides like Gantrisin and Trimethoprim

3. Give FOUR (4) the names of other media that commonly used to classify Enterobacteriaciae
group in the hospital and state it principles. (6 marks)

NO MEDIA PRINCIPLE
1 MacConkey Agar MacConkey agar is a selective and differential
culture media commonly used for the isolation of
enteric Gram-negative bacteria. It is based on the
bile salt-neutral red-lactose agar of MacConkey.
Crystal violet and bile salts are incorporated in
MacConkey agar to prevent the growth of Gram-
positive bacteria and fastidious Gram-negative
bacteria, such as Neisseria and Pasteurella. Gram-
negative enteric bacteria can tolerate bile salts
because of their bile-resistant outer membrane.
Gram-negative enteric bacteria that grow on
MacConkey agar are differentiated by their ability
to ferment lactose. If the lactose is fermented by
the bacteria, the production of the acid drops the
pH of the media. The drop in pH is indicated by
the change of neutral red indictor to pink (Neutral
read appears pink at pH’s below 6.8).
 Strongly lactose fermenting bacteria produce
sufficient acid which causes precipitation of the
bile salts around the growth. It appears as a pink
halo surrounding individual colonies or areas of
confluent growth. Pink halo in not seen around
the colonies of weaker lactose fermenting
bacteria.Gram-negative bacteria that grow on
MacConkey agar but do not ferment lactose
appear colorless on the medium and the agar
surrounding the bacteria remains relatively
transparent.

2 TCBS agar TCBS Agar is used for the selective isolation of

Vibrio cholerae and other enteropathogenic
vibrios. Thiosulfate and sodium citrate, as well as
the alkalinity of the medium, considerably inhibit
the growth of Enterobacteria. Ox bile and sodium
cholate slow the growth of enterococci and inhibit
the development of Gram-positive bacteria. The
acidification of the medium resulting from the
fermentation of sucrose by Vibrio makes
bromthymol blue turn yellow. Bromthymol Blue
and Thymol Blue are pH indicators. Using
thiosulfate as a sulfur source, the production of
hydrogen sulfide is visualized in the presence of
ferric citrate. Yeast extract and peptone provides
the nitrogen, vitamins, and amino acids in TCBS
Agar. Sodium chloride provide optimum growth
and metabolic activity of halophilic Vibrio spp.
Agar is a Solidifying agent.

An increased pH is used to enhance growth of

Vibrio cholerae, because this organism is
sensitive to acid environments.

3 Salmonella-Shigella (SS) agar Salmonella-Shigella (SS) agar is a selective and

differential medium . It is used for the isolation,
cultivation and differentiation of gram-negative
enteric microorganisms isolated from both
clinical and non-clinical specimens such as from
feces, urine, and suspected food items (fresh and
canned foods). This medium is not recommended
for the primary isolation of Shigella as some of
Shigella strains may not grow on SS agar due to
relatively high level of selectivity. The presence
of bile salts mixture and dyes (brilliant green)
inhibits the growth of gram-positive species to a
 varying degree. Differentiation of enteric
organisms is achieved by the incorporation of
lactose in the medium. Organisms which ferment
lactose produce acid which, in the presence of the
neutral red indicator, results in the formation of
red/pink colonies. Lactose non-fermenters form
colorless colonies. The latter group contains the
majority of the intestinal pathogens, including
Salmonella and Shigella.The sodium thiosulfate
and ferric citrate enable the detection of hydrogen
sulfide production as evidenced by colonies with
black centers.

4 CLED Agar CLED Agar is an abbreviation for Cystine

Lactose Electrolyte-Deficient Agar. It is a type of
differential medium recommended for diagnostic
urinary bacteriology. The medium supports the
growth of all urinary potential pathogens and
provides distinct colony morphology. CLED Agar
also supports the growth of a number of
contaminants such as diphtheroids, lactobacilli,
and micrococci. It is electrolyte deficient to
prevent the swarming of Proteus species. The
nutrients in BD CLED Agar are supplied by the
gelatin and casein peptones, and beef extract.
Lactose is included to provide an energy source
for organisms capable of utilizing it by a
fermentative mechanism. Bromthymol blue is
used as a pH indicator to differentiate lactose
fermenters from lactose-nonfermenters.
Organisms which ferment lactose will lower the
pH and
change the color of the medium from green to
yellow. The cystine permits the growth of "dwarf
colony" coliforms.3 Electrolyte sources are
reduced in order to minimize the swarming of
Proteus
species. Thus, the medium allows quantitative
determination of urinary pathogens including
Proteus when calibated loops are used for
inoculation