Laboratory Report

School of Food Science and Environmental Health Laboratory Report.

Module Code and Title: Pharmaceutical Microbiology TFMB2002

Lecturer: Swarna Jaiswal

Experiment Title: The Staphylococcus Genus

Experiment Date: 30/09/2019

Submission Date: 30/10/2019

Nadejda Sitisco
Student name(s) and number:
D19124106
1. Aim and Objectives
1.1 Aim: The aim of the experiment was to become familiar with the identity of
Staphylococcus spp., as well as to carry out the standard microbiological methods.

1.2 Objective : The objective of the experiment was to examine and compare the two
Staphylococcus spp (S. aureus and S. epidermidis), according to their morphology and
biochemical characteristics, by using standard microbiological methods such as: gram stain,
catalase test, coagulase test, TSA, MSA and DNase.

2. Introduction

The species

Two species are commonly associated with staphylococcal diseases in humans:

1. Staphylococcus aureus
More virulent strain
Some people are carriers in nose and on skin
2. Staphylococcus epidermidis
Normal microbiota of human skin
Can cause infections
Staphylococcus species are Gram positive, non-motile, non sporing cocci of varying
size occurring singly, in pars and in irregular clusters. Colonies are opaque and may be white
or cream and occasionally yellow or orange. The optimum growth temperature is 30°C - 37°C.
They are facultative anaerobes and have a fermentative metabolism .
Staphylococci are frequently known as agents of infectious processes.

S. aureus and S. epidermidis are the most important causative agents of acute and
chronic infections in humans as well as in animals.

S. aureus being the most important human pathogen. S. aureus causes superficial and
deep skin and soft tissue infections, gastroenteritis, staphylococcal scalded skin syndrome, and
toxic shock.

It is estimated that up to half of all adults are colonized, and approximately 15% of the
population persistently carry S. aureus in the anterior nares. Some populations tend to have
higher rates of S. aureus colonization (up to 80%), such as health care workers, persons who
use needles on a regular basis (i.e., diabetics and intravenous (IV) drug users), hospitalized

2
patients, and immunocompromised individuals. S. aureus can be transmitted person-to-person
by direct contact

S. epidermidis is associated with infections such as osteomyelitis, wound infections,

associated peritonitis, and nosocomial bacteraemia. Staphylococcus epidermidis, is a less
virulent than Staphylococcus aureus, is a frequent cause of infection in hospitalised patients,
often associated with implanted medical devices.

Multiple antibiotic resistance is increasingly common in S aureus and S epidermidis.

Methicillin resistance is indicative of multiple resistance. Methicillin-resistant S
aureus (MRSA) causes outbreaks in hospitals and can be epidemic.

3. Materials and Methods

List of materials

 Culture of S. aureus and S. epiderdimis

 TSA (Tryptic Soy Agar) media
 MSA (Mannitol Salt Agar) media
 DNase
 Reagents: Crystal violet (primary stain), Iodine solution/Gram's Iodine (mordant
that fixes crystal violet to cell wall), Decolorizer (ethanol), Safranin (secondary
stain)
 Water
 Reagent of ID colour Catalase (ID-ASE)
 Staphylase test kit
 Inoculation Loop
 Bunsen burner
 Marking pen
 Microscope

3
3.1. Gram Staining Procedure

Gram staining method, the most important procedure in Microbiology, was developed
by Danish physician Hans Christian Gram in 1884. Gram staining is still the cornerstone of
bacterial identification and taxonomic division.

This differential staining procedure separates most bacteria into two groups based on
cell wall composition:

1. Gram-positive bacteria (thick layer of peptidoglycan-90% of cell wall)- stains purple

2. Gram-negative bacteria (thin layer of peptidoglycan-10% of cell wall and high lipid
content) –stains red/pink

Procedure

Smear Preparation: the material was fixed on a slide with drop of water and then was heat
fixed. The slide was allowed to cool to the touch before applying the stain.

1. On the slide was applied crystal violet staining reagent.

2. Slide was washed in a gentle and indirect stream of tap water for 2 seconds.

3. On the slide was added mordant: Gram’s iodine. 1 minute for waiting
4. Slide was washed in a gentle and indirect stream of tap water for 2 seconds.

5. On the slide was applied decolorizing agent (Acetone-alcohol decolorizer). 10-15

seconds for waiting.
6. On the slide was applied a counterstain, safranin. 30 seconds to 1 minute for waiting.
7. Slide was washed in a gentile and indirect stream of tap water until no colour appears in
the effluent and then the slide was blot dry with absorbent paper.

8. The results were observed of this procedure under oil immersion (100x) using a Bright
field microscope.

4
Fig 1: Procedure of Gram Staining Fig 2: Cell wall of Gram Positive and Gram-
Negative Bacteria.

3.2 Catalase test

The catalase test is based on the ability of microorganisms to produce catalase.

Catalase is common enzyme found nearly in all living organisms that are exposed to
oxygen. During aerobic respiration, microorganisms produce hydrogen peroxide.

The catalase test is used to determine the ability of microorganisms to degrade H2O2
into Oxygen and Water. The catalase test was carried out for S. aureus and S. epidermidis as
well.

Catalase
2 H2O2 2 H2O + O2

Procedure

1. A drop of 3% H2O2 was placed on to the clean slide and mix with sterile wooden stick
2. A small amount of bacterial colony was transferred to a surface of clean slide using a
loop or sterile stick.

3. The results were observed and recorded.

5
 3.3. Coagulase test

Coagulases are enzymes that clot blood plasma by a mechanism that is like normal
clotting. The coagulase test identifies whether an organism produces this exoenzyme. Bound
coagulase otherwise known as a Clumping factor can be detected by carrying out a slide
coagulase test.
This test evaluates for the presence of coagulase, an enzyme that coagulates blood
plasma, and can differentiate between Staphylococcus aureus and Staphylococcus epidermidis.

Coagulase Coagulase reacting factor

Prothrombin like activity

Fibrinogen Fibrin

The test also indicates the ability of an organism to clot plasma by the action of the
enzyme coagulase.

Procedure

1. Using a Microgen® Staph Latex Agglutination Kit, a drop of coagulase reagent was added
on a clean slide, for both section of species.
2. Using a sterile stick the bacterial colony of S. aureus and S. epidermidis was collected.
3. The bacterial colonies are transferred and mixed with coagulase reagent.
4. The results was observed and recorded.

6
3.5. TSA (Tryptic Soy Agar), MSA (Mannitol Salt Agar) DNase

3.5.1. TSA (Tryptic Soy Agar)

Tryptic Soy Agar is a universal media which supports the growth of variety of
microorganisms which means that both gram positive as and gram-negative bacteria grow in
this medium. TSA nonselective media providing enough nutrients to allow for a wide variety
of microorganisms to grow.
Tryptic Soy Agar (TSA) is mainly used as an initial growth medium for the purposes
of observing colony morphology and for biochemical testing and culture storage. Tryptic
Soy Agar contains digests of soybean meal and casein, making it suitable for the growth of a
wide variety of microorganisms.
Tryptic Soy Agar (TSA) is recommended for use as a general growth medium for the
isolation and cultivation of microorganisms.

3.5.2. MSA (Mannitol Salt Agar)

Mannitol Salt Agar is a selective media which allows growth of some organisms while
inhibiting growth of others. MSA allows to grow of Staphylococcus species and species in the
family Micrococcaceae but high salt concentration inhibit other bacteria.
Mannitol Salt Agar is differential media that visually distinguish species base on biochemical
characteristics:
- Contains mannitol and the pH indicator phenol red which gives to MSA characteristic
bright pink colour to detect acid from fermentation, resulting in yellow zone around the
colonies

7
3.5.3. DNase
DNase test is used to determine the ability of an organism to hydrolyse DNA and
spend it as a source of carbon and energy for growth. The DNase test is particularly useful
when the results of a coagulase test are difficult to interpret.
DNase Agar is used primarily in clinical laboratories to differentiate Staphylococcus aureus
from other Staphylococci based on deoxyribonuclease activity.

Procedure of streaking on TSA, MSA and DNase media

1. The plates was divided in two and labelled with the initial, bacterial strain and media type
2. The loop was heated, and the culture of S. aureus and S. epidermidis was collected with
sterile loop.
3 The streaking was done on the TSA, MSA and DNase culture media.
3. The streaking plates was placed in incubator for 24 h, at 37°C.

4. Results
Gram stain (S. aureus and S. epidermidis)

S. aureus are cocci that form irregular grape-like clusters, the colonies are 2-3 mm in
diameter. They are non-motile and non-sporing. They rapidly grow and abundantly under
aerobic conditions. The golden appearance is the etymological root of the bacterium's
name, aureus means golden in Latin.
S. epidermidis are approximately 0.5 to 1.5 nm in diameter and arranged in grape- like
clusters. They are facultative anaerobes that can grow by aerobic respiration or by fermentation.
Both bacteria are gram positive after Gram Stain procedure was done and the cultures
was examined under Oil Immersion (100X) lens.

8
 S.aureus (Gram +ve cocci) S. epidermidis (Gram +ve cocci)

Fig3. Morphology and Gram stain

Microbe Colour
Gram Cell Size of the
Arrangement Elevation of the
result shape Colony
Colony

Staphylococcus Positive Coccus Clustered Flat

White
small
aureus yellow

Staphylococcus Negative Rod Clustered Flat

Off
small
epidermidis white

9
 Catalase results

Catalase positive reaction is meant to cause bubbles while catalase negative reaction
is not destinated to produce any bubbles at all. Catalase test is a particularly important test
used to determine whether the gram-positive cocci is a Staphylococci. S. aureus and
S.epidermidis are both catalase positive.

Coagulase results

The coagulase test is one way to differentiate the highly pathogenic S. aureus from the
other less pathogenic staphylococcal species on the human body. S. aureus is a coagulase
positive organism while S. epidermidis is coagulase negative.

S. aureus S. epidermidis

10
Fig. 4 Table of results

S. aureus S. epidermidis

Gram stain Gram +ve cocci Gram +ve cocci

Coagulase Positive Negative
Catalase Positive Positive
TSA Light, gold colonies White, consistent colonies
MSA Ferment mannitol Does not ferment
DNase Positive Negative

MSA

MSA is differential and selective medium. MSA is selective because it contains 7.5%
salt–a high salt concentration that promotes the growth of some organisms but can inhibit the
growth of others.
MSA is a differential medium because it contains the sugar mannitol and the pH
indicator phenol red. Organisms that can ferment mannitol produce acid by-products, causing
a colour change. Phenol red is a cherry red colour above pH 8.5, yellow red from pH 6.9 to 8.5,
and bright yellow at pH 6.9 or lower.
Both Staphylococcus epidermidis and Staphylococcus aureus can tolerate the high salt
content of MSA. S. aureus can ferment mannitol, which turns the colour of the phenol red
indicator present in the medium from red to yellow.
S. aureus colonies was surrounded by yellow zones, while S. epidermidis cannot
ferment mannitol and consequently no changes of red medium occur. Mannitol Salt agar helps
to differentiate between S. aureus from other species of Staphylococcus.

11
 TSA

The result was recorded and analysed: was observed that S. aureus produces the yellow
pigment staphyloxanthin and characteristic light gold coloured colonies are formed on all rich
media including Tryptic Soy agar (TSA) at 37°C. S. epidermidis forms white, consistent
colonies on Tryptic Soy agar.
Tryptic Soy agar (TSA) is a basic nutrient media that it will support the grow of S.
aureus and S. epidermidis.

DNase

DNase Agar found uses mostly in clinical laboratories in order to differentiate

Staphylococcus aureus from other Staphylococci based on deoxyribonuclease activity. The
media contains DNA, and when the surface of the medium is covered with a small quantity of
1N Hydrochloric Acid, the acid precipitates the DNA.
Staphylococcus aureus produce DNase enzymes that hydrolyse DNA resulting in a clear area
around the colonies, described as being DNase positive; while S. epidermidis do not produce
clearing.

Fig 5. TSA, MSA and DNase results

DNase MSA TSA

12
 5. Discussion
The principle of experiment was to become familiar with the identity of Staphylococcus
spp., as well as to carry out the standard microbiological methods. During the experiment was
compared two Staphylococcus spp. (S. aureus and S. epidermidis), according to their
morphology and biochemical characteristics, by using standard microbiological methods such
as: gram stain, catalase test, coagulase test, TSA, MSA and DNase. The results that was
recorded are successful complete and met expectation.

Was noted that both Staphylococcus spp. are Gram-positive species. Was observed
that the clusters arise because staphylococci divide in two planes. This clustering helps to
distinguish staphylococci from streptococci, which usually grow in chains.

Among all test, was carried out catalase and coagulase tests.

Catalase test is primarily used to distinguish among Gram-positive cocci: members

of the genus Staphylococcus that are catalase positive, while the members of the genera
Streptococcus and Enterococcus are catalase negative.

Coagulase test is used to differentiate Staphylococcus aureus (positive)

from Coagulase Negative Staphylococcus. The coagulase-positive staphylococci constitute
the most pathogenic species S aureus. which is known to produce enterotoxin. The test and
results of coagulase production by suspected food-poisoning isolates is one of the most
important properties tested for. Coagulase-negative staphylococci present less harm on skin but
some of these may cause infections as well.

As well during the experiment the results was recorded by analysing plates of MSA,
TSA and DNase:

 MSA (Mannitol Salt agar) is differential and selective medium. MSA is

selective media contain 7.5 % salt - high salt concentration that inhibit the growth of
some organisms but allow others to grow. In our case mannitol salt agar, which contains
a high concentration of sodium chloride inhibits the growth of most organisms but
permits Staphylococci to grow.

13
 TSA (Tryptic Soy Agar)
TSA is used for the isolation and cultivation of nonfastidious and fastidious
microorganisms.
An example of nonfastidious bacteria is the Staphylococcus species.
Staphylococcus species can grow in nutrient-sparse environments and can survive in a
wider temperature range than many fastidious bacteria.
TSA is used for the isolation and cultivation of large variety of
microorganisms. Tryptic Soy Agar (TSA) is mainly used as an initial growth medium
for the purposes of observing colony morphology, developing a pure culture and
achieving sufficient growth for further biochemical testing and culture storage.

 DNase
DNase test is performed to find out the organism’s ability to hydrolyse DNA
and use it as a carbon source, which will in turn use as energy for the organism to grow.
The primary use of the DNase test is to differentiate S. aureus which is DNase
positive so it can produce Deoxyribonuclease enzyme from other Staphylococci that do
not produce enzyme DNase which is S. epidermidis present as the analysing bacteria in
the experiment.

14
 Bibliography
1. Anon., 2019. Deoxyribonuclease (DNase) Test – Principle, Procedure, Uses, Result
Interpretation. [Online]
Available at: https://laboratoryinfo.com/deoxyribonuclease-dnase-test/
[Accessed 29 October 2019].
2. Anon., 2013. Catalase test: Principle, Procedure, Results and Applications. [Online]
Available at: https://microbeonline.com/catalase-test-principle-uses-procedure-results/
[Accessed 27 October 2019].
3. Aryal, S., 2019. Mannitol Salt Agar for the isolation of Staphylococcus aureus.
[Online]
Available at: https://microbiologyinfo.com/mannitol-salt-agar-for-the-isolation-of-
staphylococcus-aureus/
[Accessed 28 October 2019].
4. Hogg, S., 2005. Essential Microbiology. Chichesster, West Sussex: John Wiley &
Sons, Ltd.
5. Howden, B., n.d. Staphylococcus aureus and Staphylococcus epidermidis. [Online]
Available at: https://biomedicalsciences.unimelb.edu.au/sbs-research-
groups/microbiology-and-immunology-research/molecular-epidemiology,-genomics-
and-pathogenesis-of-antimicrobial-resistant-bacteria/staphylococcus-aureus-and-
staphylococcus-epidermidis
[Accessed 28 October 2019].
6. Madigan, M. T. et al., 2019. Brock Biology of Microorganisms, Global Edition. 15th
ed. s.l.:Pearson (Intl).
7. Marin Schweizer, L. H., n.d. Hospital Infection Control Gram positive bacteria –
Staphylococcus aureus. [Online]
Available at: https://www.infectiousdiseaseadvisor.com/home/decision-support-in-
medicine/hospital-infection-control/gram-positive-bacteria-staphylococcus-aureus/
[Accessed 28 October 2019].
8. NHS, 2014. UK Standards for Microbiology Investigations. [Online]
Available at:
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachme
nt_data/file/827627/ID_7i3.pdf
[Accessed 28 October 2019].
9. S. P Denyer, W. B. H., 2011. Hugo and Russell's Pharmaceutical Microbiology. 8th
ed. London: Wiley-Blackwell.
10. Tulane University, S. o. M., 2016. Bacteria 101: Cell Walls, Gram Staining, Common
Pathogens. [Online]
Available at:
http://tmedweb.tulane.edu/pharmwiki/doku.php/bacteria_101_cell_walls_gram_staini
ng_common_pathogens
[Accessed 28 October 2019].

15
16