Sultan Qaboos University

Collage of Agriculture and Marine Science

Department of Animal and Veterinary Science

Diagnostic Microbiology (ANVS4213)

Diagnostic Microbiology Lab 7:

Unknown Sample Identification

Done by:

Abdullah Bin Said Al Rab’Ani - 128300

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Introduction:
A three-year old ewe was presented with a history of enlarged submandibular lymph node. A
sample from the abscess was collected and sent to the lab to determine the causative agent of the
case and the appropriate treatment.

Methods & Materials:

Direct Gram stain: doing of Gram stain to determine if the sample is Gram-positive or Gram-
negative. In addition, to facilitate study of the bacterial morphology under the microscope. This
is done twice, once before and after culturing.

Figure 1: Gram staining steps Figure 2: Bacterial morphology

KOH test: a rapid test to distinguish between Gram-positive and Gram-negative bacteria. by
placing one drop on a slide of potassium hydroxide and spread bacteria on it. In negative gram
bacteria a sticky mucus threads will observe. If nothing is observed this mean that its Gram-
positive bacteria. The reason for that is broken down of the thin wall layer of peptidoglycan present
in Gram-negative bacteria and release of viscid chromosomal material.

Catalase: rapid test of hydrogen peroxide (H2O2) molecule aid to differentiate between
Streptococcus and Staphylococcus, by broken down H2O2 and form H2O + O2(gas). The appearance
of bubbles is evidence of catalase enzyme production and this indicate it is staphylococcus.

Oxidase test: the use of rapid oxidase test to know if the bacteria is enteric or not. This done by
using oxidase strip that contain for tetramethyl-p-phenylenediamine, the change of this substrate
to indophenol dark blue is result of oxidase enzyme production.

TSI agar: this agar used to determine if the negative gram bacteria able to ferment sugar (glucose,
lactose, sucrose) and produce hydrogen sulfide (H2S). In addition of bacterial ability to utilize

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peptone (nitrogen source), and this agar contain of phenol red that work as indicator for CO2 or H2
production. This agar should be curved while preparing to provide aerobic medium to know if the
bacteria is aerobic or not. The Sample inoculated with a needle at the bottom and streaked at the
slant and left to incubate for 18-24hrs at 35 °C. Change of original orange Colour indicate sugar
fermentation, change into red means the bacteria are able to utilize peptone, agar splitting is reason
for gas production, and black precipitate is a reason for H2S production.

API: a strip that contains dehydrated test tubes that are filled with a medium made by inoculating
multiple colonies in suspension medium. And after incubation of this strip for 24hrsr at 37°C. some
solutions added to read the strip and then use a program for reading the API. Chosing of API test
depend on several test (catalse, oxidase, and cell morhplogy)

AST: this test measures the ability of antibiotics to inhibit microbial growth, and this to determine
the more suitable treatment against this type of bacteria. we use Muller-Hinton agar. These bacteria
mixed with special saline and measured by Diluhpotometer device that measure the turbidity of
solution. The turbidity of solution should reach about 0.5, then spread the solution on agar using
swap or any material facilitate spreading. Then the antibiotics should be placed far from each other
to get right reading, and incubate it for 24hrs at 37°C.

Result & Discussion:

This bacterium has a greyish small round colony and it’s not able to cause hemolysis in blood agar
(Gamma). After staining, the bacteria under the microscope were looks between cocci and bacilli
which indicate that was coccobacilli. The KOH and oxidase were negative, but the catalase was
positive, this mean that this bacterium is gram + and its able to produce catalase enzyme and cause
bubbles formation. According to the morphology and pervious results this could be a coryne
bacteria. TSI agar was (k/a) this mean that bacteria ferment 0.1% glucose and utilize the peptone

Figure 3: Gamma Hemolysis Figure 4: TSI result (K/A)

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presented on red colour of slant, and the rest of 1% of sucrose and lactose are not fermented. The
API was Corynebacterium diphtheriae biotype gravis by 98.2%.

Figure 5: API result

Next table show the diameter measurement for antibiotics sensitivity. All antibiotics has the ability
to inhibit growth of this bacteria Antibiotic Code Zone Interpretation
but the RA-30 has the highest diameter
(mm)
diameter which makes it the best
Kanamycin K-30 39 Susceptible
antibiotic.
Erythromycin E-15 51 Susceptible
Rifampicin RA-30 55 Susceptible
Tetracycline Te-30 41 Susceptible
Conclusion: Table 1: Antibiotics Result

The purpose of all these steps and result is to identify the causative agent for lymph node
enlargement present on that ewe. The causative agent founded was Corynebacterium diphtheriae
biotype gravis and this bacterium is makes toxins can lead to several animals or humans’ problem
and one of the signs include swollen of gland (enlargement of lymph node). And using any of these
antibiotics can eliminate the pathogen.

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