GRAM POSITIVE BACTERIA

Gram positive

(violet/purple color)

-Spherical (cocci) in shape (in general)

-Chain like in arrangement (in general)

Gram positive (violet/ purple color)-spherical (cocci) in shape -clusters in arrangement

We obtain many of the gram positives from throat swabs

we culture gram positive bacteria on:→ Blood or→ CLED (enrichment media)

Next, we’ll learn how to differentiate between the staphs we’re interested in by there looks on both media.

BLOOD AGAR

A- Staphylococcus albus

Also called Staph. epidermidis

When it grows on blood, it gives white (albino) colonies.

B- Staphylococcus aureus

When it grows on blood, its colonies gain a golden color.

CLED media Lactose fermentation ( +ve )

staphylococcus spp.

CLED differentiates between the two by lactose fermentation, aureus ferments lactose turning CLED color to this golden you can see above, albus also gives a golden whitish color. So, BLOOD agar is the better differentiator.

TEST FOR DIFFERENTIATION OF STAPHYLOCOCCUS SPECIES

After we culture bacteria on blood or CLED, we re-stain it by gram stain to make sure it’s Staph. not Strep.. After we confirm, we do the following tests to differentiate between species of staphylococci.

Catalase distinguishes all Staphylococci from Streptococci.All Staphylococciare catalase positive, while Streptococci aren’t

Mannitol Salt Agar and Coagulase test differentiate between Staph. aureus and Staph. albus

CATALASE TESTCatalase is an enzyme, We test its presence in the bacteria.Staphylococci are catalase positiveStreptococci are catalase negative How does it work?Catalase breaks H2O2

into H2O and O2, We put a drop of H2O2 on a slide, then we put some of the bacterial colony on it.If gas bubbles are formed, it means catalase is present.

MSAMANNITOL SALT AGAR MEDIA (DIFFERENTIAL)

Studies fermentation of mannitol.between aureus and albus.We purely culture the bacteria on it, and the next day we observe Golden = aureus (mannitol +ve)

White colonies, same media color = albus(Mannitol fermentation –ve)

S.aureus

S.albus

Between aureus and albus. How?we put plasma in a tube then we

add bacteria, if:

A clot forms it means we have

No clot forms, it means we have

*Hemolysis is RBCs break down that makes them lose their red color.*

We see colonies as pen point colonies

Mainly we group them by their action of hemolysis.

Streptococcus mutans Enterococcus faecalis

Further explanation of hemolysis is present in sheet 12 microbiology.don’t memorize the names of the bacteria on the pictures above.

We have a bacteria with greenish hemolysis (alpha hemolytic), then we need to find out if it’s pneumonia or viridans.

To differentiate between pneumonia and viridans we use a test called optochin test. It’s a sensitivity test towards an antibiotic called optochin.How it’s done?If we found an alpha hemolytic bacteria, we culture it then we put an optochin disk on the media.

The next day we observe, if there’s an inhibition zone (no bacterial growth) that is at least 14 mm in diameter, then we say the bacteria is optochin sensitive and we classify it as pneumonia. If there’s no or an inhibition zone that is less than 14 mm, we say this bacteria is optochin resistant and we classify it as a viridans group member.

Notice here, there’s no clearance in the greenish area, it’s still fully green. Unlike the above picture.

STREPTOCOCCUS PNEUMONIAE

Strep. Pneumonia has a different arrangement than the general chain like arrangement, it’s called lancet shaped (like glasses or like 8)

STREPTOCOCCUS VIRIDANS

Their arrangement is in chains but their shape is coccobacilli

Differentiation between beta hemolytic Strep.

We use a test called bacitracin test.Here, diameter doesn’t matter.Sensitive in any diameter means it’s pyogenes.Resistant means agalactiae

agalactiae pyogenes

Gamma hemolysis

streptococcus

Enterococcus

Group D

- E.Faecalis

Other than Enterococcus

group D

We use bile esculin test

Enterococcus

Group D

Positive

Other than

Enterococcus

Group D

Negative

We pour bile-esculin media (differential media) in a tube, then we put the bacteria and leave it for a day. if the media turns into black then the bacteria is bile esculin positive and we classify it as Enterococcus.Otherwise we say it’s other than enterococcus.

Bile is a high salt concentration solution.Esculin is an iron derivative, a reaction by it turns the media into a black color when the test is positive.

STREPTOCOCCUS PNEUMONIAE

➢Gram +ve cocci

➢Short chain

➢Commonly in

pairs, but can be in

short chains too.

➢Lancet shape

•Gram positive cocci

•Short chain

•Diplococcus

Enterococcus

Gram Positive

Cocco-bacilli

Arrangement as

Chinese letter

Diphtheroids

BACILLUS SPP.We culture it on blood.It comes in large, dry colonies with irregular edges.And it forms spores.

BACILLUS SPP.

These are spores, they don’t stain with gram stain, we use other stains if we want to stain them.

And these are the gram positive bacillus chains.

BACILLUS CEREUS