MICROBIOLOGY LABORATORY 4 Gram stain of pus from cervix – Gonorrhea

USTMED ’07 Sec C – AsM; Photos provided by JV.N & MeaM.

Culture of Microorganisms

Different Streaking Methods

Culture Media Used: Eosin Methylene Blue

Organisms Used: Escherichia Coli
Method of Streaking: Simple Streaking
Neisseria gonorrhea – Gram stain
Illustration:

Neisseria gonorrhea –
Stanford’s stain
Culture Media Used: Eosin Methylene Blue
Organisms Used: Escherichia Coli
Method of Streaking: 4 Quadrant Method of Streaking

Note: This method of streaking is used for better isolation of

the organism

Illustration:

Clinical specimen: Urethral Discharge

Staining used: Gram Staining
Results: Gram Negative Cocci in Pairs Intracellularly located

Culture Media Used: Eosin Methylene Blue

Organisms Used: Escherichia Coli
Method of Streaking: Overlapping method of streaking

Note: This method of streaking is used for sensitivity testing

Illustration: Staphylococcus aureus

Gram Positive Cocci in clusters

Catalase Test – Positive

(best single test for the pathogenecity of Staph. Aureus)

Colonies on plated media:

1. Blood agar plate (BAP) – Beta hemolytic (complete

hemolysis)

2. Mannitol salt agar (MSA) – Yellow colonies (due to

mannitol fermentation)

Demo slides:
Gram stain of pus from an epyema cavity showing long and
short chains of Gram-positive streptococci and large numbers
of pus cells (stained red) x3,900.
Staphylococcus aureus on mannitol Salt Agar (MSA),
differentiating S. aureus from other catalase positive gram
posititve cocci. The medium contains 7.5% NaCl which inhibits
the growth of many organisms. S. aureus ferments mannitol
resulting to acid end product causing the phenol red indicator
to change from pink to yellow.

Staphylococcus aureus. Gram stain of culture showing

characteristic irregular clusters of gram-positive cocci. There
are no spores or capsules.
Staphylococcus aureus on Mannitol Salt Agar (MSA) Staphylococcus aureus on BAP
Showing yellow colonies due to Mannitol fermentation colonies showing beta or
complete type of hemolysis.

Mannitol Salt Agar (MSA) differentiates S. aureus from other Growth of staphylococcus on
catalase-positive gram-positive cocci. The medium contains BAP with characteristic
7.5%NaCl which inhibits the growth of many orgs. pigmentation (golden-yellow)
and ‘oil paint’ appearance.

Streptococcus pneumoniae
(+) results – fermentation of mannitol resulting to acid end
product causes the phenol red indicator to change from pink to - gram positive cocci in pairs
yellow (right half of the plate) - catalase negative
- alpha hemolytic/incomplete hemolysis on BAP
(-) result – no fermentation, no chage of color (left half) - Sensitive to Optochin Disk Test as seen in Chocolate
Agar Plate (CAP)

Streptococcus viridans
- gram positive cocci in chain
- catalase negative
- alpha hemolytic/incomplete hemolysis on BAP
- Resistant to OPtochin Disk Test as seen in CAP

Streptococcus pyogenes
- gram positive cocci in chains
Colonies of Staph. Aureus on MSA (close-up). Medium - catalase negative
surrounding colonies has turned yellow - beta hemolytic/complete hemolysis on BAP
- resistant to optochin disk
Coagulase test confirms identiy as Staphylococcus aureus
(coagulase positive) and Staphylococcus epidermidis (coagulase Optochin sensitivity of
negative): pneumococci in contrast with
Streptococcus viridans which
1. Slide coagulase test – when a saline suspension of also produces alpha-
staphylococci is mixed with human or rabbit plasma haemolytic colonies on blood
on a slide, there is immediate clumping with agar.
coagulase positive staphylococci.

Streptococcus viridans on BAP

- produces alpha hemolytic/incomplete hemolysis on
BAP

2. Tube coagulase test – when diluted plasma is

inoculated with S. aureus, jellification occurs w/in 2-
4 hours. This result is due to coagulase converting
fibrinogen in the plasma to fibrin and a positive test
confirms a positive slide test.

Growth of a beta-haemolitic
stretptococcus on blood agar
medium showing large zones
of clear haemolysis around
small transparent colonies.

Staphylococcus aureus on Blood Agar Plate (BAP) Growth of pneumococci on blood agar showing characteristic
Showing beta hemolytic hemolysis; complete hemolysis ‘draughtsman’ colonies with concentric surface rings.
Colonies of group A Strep on 5% sheep blood agar. Colonies of
group A Streptococci on 5% sheep blood agar are small and
surrounded by wide zones of beta hemolysis.

Gram stain of Streptococci in broth culture (x1250). Gram stain

of a positive blood culture broth demonstrating gram-positive
cocci arranged in chains. Streptococci are normal microbiota
in the upper respiratory tract and the gastrointestinal tract.
For this reason, gram stains of specimens from these sites are
not helpful in diagnosing infections caused by the pathogenic
Streptococci such as S. pyogenes and S. pneumoniae.

Neisseria Gonorrhea

Gram stain for N. Gonorrhea

Culture of N. gonorrhea on selective media (35oC in 5% carbon

dioxide environment)

- fin -

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