VMCB 55
VETERINARY MICROBIOLOGY AND BACTERIOLOGY
EXERCISE 1
ISOLATION, CHARACTERIZATION AND ANTIBIOTIC SENSITIVITY TESTING OF
Staphylococcus spp. FROM VARIOUS SAMPLES
LABORATORY III
GROUP 5
CANEDO, ABEGAIL
ESGUERRA, NOEL
GALILA, JOREN
GAYARES, GIDEON
MAGPANTAY, FRANCES NICOLE
FEBRUARY 13, 2019
I. ABSTRACT
Staphylococci are generally catalase - positive and are usually categorized by
their colonial appearance, haemolytic pattern, biochemical profiles and ribosomal
RNA gene restriction patterns (Thomson - Carter et al., 1989). Coagulase - negative
staphylococci exhibit variation in their ability to produce haemolysis which usually
develops slowly. This experiment is aimed to isolate and determine pure cultures of
the bacteria using different isolation techniques. Samples from different sources;
nose, fomite and unknown-control, first undergo to the Gram-staining technique to
identify whether the samples are possible staphylococcus species. Samples are
inoculated into nutrient broth and then into nutrient agar slant for culture. Mannitol
salt agar was used as a selective-differential media. Cultures grown in this media are
further tested for their ability to utilize different carbohydrates and production of gas
using mannose, sucrose, dextrose, lactose, sorbitol, and maltose. Also, the samples
are inoculated into Muller-Hinton Agar for antibiotic sensitivity testing.
II. INTRODUCTION
Staphylococcus (Staphyle, in Greek, meaning ‘bunch of grapes’: kokkos,
meaning a berry) due to the typical occurrence of the cocci “in grape-like clusters.
They are spherical cocci, approximately 1 μm in diameter. Cluster formation is due to
cell division occurring in three planes, with daughter cells tending to remain in close
proximity. They may also be found singly, in pairs and in short chains of three or four
cells, especially when examined from liquid culture. They are non-spore forming,
non-motile and usually non-capsulate with the exception of rare strains. Some are
capable of haemolysis. Oxygen requirement can be anaerobic and facultative
anaerobic that causes opportunistic infections among animals. Staphylococcus sp. of
veterinary importance affects animals such as Canine, Feline, Bovine, Equine,
Porcine, Fowls, and Dolphins.
Staphylococci are gram-positive cocci. The genus Staphylococcus contains
33 defined species and 20 species found in man. It is normally found at the skin,
mucous membrane, upper respiratory tract and digestive tracts. Generally they are
also catalase positive with the exception of some strains.
In this exercise, we conducted isolation, morphological and biochemical
characterization and antibiotic sensitivity testing of Staphylococcus spp. from nasal
mucosa, watch as the fomite and an unknown culture.
II. OBJECTIVE
This exercise on “Isolation, Characterization and Antibiotic Sensitivity Testing
of Staphylococcus Spp. from Various Samples” seeks to attain the following
objectives:
1. To identify from which samples or cultures contains Staphylococcus spp.
2. To morphologically describe and differentiate the three samples.
3. To characterize biochemically the colonies of three different samples.
4. To isolate a pure culture of Staphylococcus spp.
5. To determine the antibiotic sensitivity profile of the three different isolates.
III. MATERIALS
Samples of culture from nasal mucosa, watch as fomite, and an unknown culture
Nutrient broth
Agar slant
Sterile cotton swab
Hydrogen peroxide (10 vol)
Mannitol Salt Agar plate
Carbohydrate media- dextrose, lactose, maltose, sorbitol, sucrose
Sterile water
IV. METHODOLOGY
Specimen collection
Three different samples were collected and labelled as nose, fomite, and an
unknown-control. First, the unknown-control was inoculated in a nutrient broth and
set aside. Sterile cotton swabs are used to collect the two other samples. The swab
was partially immersed to the nutrient broth for it to moisten and swab it to the nasal
membrane just inside the nostrils. The same procedure was applied to our fomite.
After transferring all samples in the nutrient broth, the tubes were incubated at 37°C
for 24 hours.
Isolation and Evaluation
After 24 hours of incubation, turbidity was observed in the broth cultures; the
organisms were transferred aseptically from nutrient broth to nutrient agar slant and
incubated it again at 37°C for another 24 hours.
The organisms grow from the nutrient agar slants; the cultures from the slants
were inoculated to a mannitol salt agar (MSA) which was a selective media. The
plate was divided into three parts. Each of the three samples was streaked over the
agar surface with a quadrant streak to make sure it thins out and get the pure culture.
The plate was incubated aerobically at 37°C for 24 hours. We observed the colony
and characterized it.
Morphological Characterization
The inoculum from the agar slant was collected and used to prepare the gram
staining technique to identify whether it belongs to a gram positive or gram negative
organisms. The fixed smear from the three samples of the presumptive
Staphylococcus organism were prepared; in this technique Crystal violet was used as
primary stain for 1 minute, Lugol’s iodine for 1 minute as mordant, acetone alcohol
for 10 seconds as decolorizer and Saffranin for 30 seconds as counter stain. The
slide was rinsed by water after applying every reagent. The samples were air-dried
and were viewed under the microscope.
Biochemical Characterization
From mannitol salt agar, the inoculums from three different samples were
mixed with different carbohydrate media namely maltose, lactose, dextrose, mannitol,
sorbitol and sucrose. We incubated it at 37°C for 24 hours.
Catalase test
From the nutrient agar slant, we transferred the colony to a clean grease-free
glass slide and added one to two drops of hydrogen peroxide.
Antibiotic Sensitivity Testing
Densities if the broths were compared with the prepared density standards. A
swab was taken aseptically from the broth culture. The swab was spread to the
media and let it dry. Sterile forceps was used to place the antibiotic disc in the media.
It was pressed to make sure that it has contact with the agar and has a regular
interval.
Sterile forceps was used to place the antibiotic disc at regular interval and
gently pressed it to make sure that it has contact with the agar. The plate was
incubated for 24 hours at 37°C. Zone of inhibition was interpreted and measured in
mm with the standard sizes of the zones.
V. RESULTS AND DISCUSSIONS
ISOLATION AND EVALUATION
Nutrient Agar Slant
The nutrient agar slants have a smooth glistening surface, an entire edge, a soft
consistency and an opaque, pigmented appearance. Most strains produce golden-
yellow pigment.
Fig. 1 Unknown-control culture in agar slant
Fig. 2 Fomite culture in agar slant
Fig.3 Nose culture in agar slant
Mannitol Salt Agar (MSA)
The Mannitol Salt Agar (MSA) is a selective and differential media for isolation of
Staphylococcus spp. Generally, Staphylococcus spp. can tolerate high saline
concentration that ranges from 7.5% up to 9%. In line with this, MSA is determined
as selective media because it contains high concentration of salt that is favorable for
the growth of Staphylococcus organisms. It is also a differential media because MSA
contains the sugar mannitol and the pH indicator phenol red. If an organism can
ferment mannitol, an acidic by product is formed that will cause the phenol red in the
agar to turn golden-yellow color. Most pathogenic Staphylococci, will ferment
mannitol and on the other hand, most of non-pathogenic Staphylococci will not
ferment mannitol.
Based on the results, nose shows ample amount of growth while fomite shows
minimal growth and the unknown control with no growth. It indicates that the
organisms present in the nose sample can tolerate the high salt concentration as
compare to the fomite and the unknown- control. The nose sample grows well on the
MSA which is one of the characteristics of Staphylococcus organisms.
Fig. 4 Cultured samples in Mannitol salt agar
MORPHOLOGIC CHARACTERISATION
Gram staining
The researchers performed a gram staining technique to determine whether the
bacterial samples are gram positive or gram negative. In this technique the authors
use Crystal violet for 1 minute as primary stain, Lugol’s iodine for 1 minute as
mordant, Acetone alcohol for 10 seconds as decolorizer and Saffranin for 30 second
as counter stain. The process is rinsed by water after every reagent. If the inoculum
appears purple or violet upon viewing under the microscope the result will be gram
positive bacteria but if the bacteria stains red or pink it is classified as a gram
negative bacteria. Gram positive bacteria retain the color of the primary stain
because of their thicker peptidoglycan layer that traps the crystal violet complex.
Gram negative bacteria retain the color of the secondary stain because of its thinner
peptidoglycan layer that allows the crystal violet complex escape during
decolorization.
Based on the experiment, the result shows a purple or violet color from the three
samples. This is an indicator that the three samples were gram positive bacteria that
is a characteristic of a Staphylococcus organism. However, the fomite sample also
contains other organisms which are an indication that the sample is not a pure
culture.
Fig. 5 Gram-positive species from nose
Fig, 6 Gram-positive cocci species from unknown-control
Fig. 7 Gram-positive cocci and bacillus species from fomite
BIOCHEMICAL CHARACTERIZATION
Gas production nose fomite Unknown-control
Dextrose - - -
Lactose + - -
Sorbitol - - -
Sucrose - - -
Maltose - - -
Mannose - - -
Fig. 8 Carbohydrate utilization test of nose culture
Fig. 9 Carbohydrate utilization test of unknown-control
Fig. 10 Carbohydrate utilization test of fomite culture
CATALASE TEST
A catalase enzyme neutralizes the bactericidal effects of hydrogen peroxide
and protects them. Catalase positive bacteria include strict aerobes as well as
facultative anaerobes. They all have the ability to respire using oxygen as a terminal
electron acceptor. Catalase negative bacteria may be anaerobes or they may be
facultative anaerobes that only ferment and do not respire using the oxygen as a
terminal elector receptor. Catalase enzyme cleaves the hydrogen peroxide into water
and oxygen free radicals. It is also used to differentiate the staphylococcal organism
and streptococcal organisms.
After getting a loopful inoculum from the three different nutrient agar slants,
the authors mixed it with hydrogen peroxide for catalase test. The appearance of
bubbles determines that the isolated samples are a catalase positive which is one of
the characteristics of Staphylococcus organism.
Fig. 10 Catalase test of fomite culture
Fig. 11 catalase test of nose culture
Fig. 12 Catalase test of unknown-control
ANTIBIOTIC SENSITIVITY TESTING
The antibiotic sensitivity test is used to determine if the bacterial culture is
resistant or susceptible to certain antibiotics.
ANTIBIOTIC RESULTS NORMAL
Penicilin G
Cefalexin
Fig. 13 Zone of inhibition evident in culture inoculated with culture from fomite
V. REFERENCES
Essentials of Microbiology --- Surinder Kumar MD DNB MNAMS Director
Professor Department of Microbiology Maulana Azad Medical College New
Delhi, India
Journal of Medical Microbiology --- Dr Norman Fry, Public Health England,
UK
Professor Kalai Mathee, Florida Interational University, USA
Jorgensen, J.H., et al. 2015. Manual of Clinical Microbiology, 11th ed.
American Society for Microbiology, Washington, D.C.
MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification,
Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.
Tille, P. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St.
Louis, MO.
Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III.
American Society for Microbiology, Washington, D.C.
Quality Assurance for Commercially Prepared Microbiological Culture Media,
M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS),
Wayne, PA.
Mannitol Salt Agar. Biokar Diagnostics – Rue des Quarante Mines – ZAC de
Ther – Allonne – B.P. 10245 – F60002 Beauvais Cedex – France.
