Practical 15: Pathogenic and commensal bacteria of the skin

Staphylococcus aureus

Can be pathogenic and is a common cause of skin infections

Frequently found in nosocomial infections (MRSA)

Causes mastitis in humans and animals

Can cause food poisoning through ingestion of superantigen toxin

Staphylococcal coagulase activity

Pathogenic staphylococci produce coagulase enzymes that coagulate plasma

Production of coagulase is an indicator that a strain of Staph. is S.aureus

How does a positive result appear in coagulase activity test?

Formation of fibrin that polymerises and becomes evident as a delicate veil of protein.

What is the first step of Gram staining?

First make a thin smear of them on a glass slide and then fix them to the slide

What is the most frequent errors when creating thin smear for Gram staining?

Use of too much water on slide before adding the microorganism which will make the smear too thick.

Why add water to the slide when visualising bacteria?

1. Facilitates emulsification- Bacteria on an agar plate are packed into dense, sticky colonies so if directly added onto a slide, they would remain in a thick, opaque mound.

2.Achieving a "Monolayer"- you can spread the suspension thinly across the slide. As the water evaporates, it leaves behind a thin film of bacteria that are perfectly spaced for viewing.

3.Ensures even staining- Stains (like Gram stain or Methylene Blue) cannot penetrate thick clumps of bacteria effectively.

When is water not added to slides prior to adding sample?

Films made from liquid media, from swabs or from bodily fluids are spread directly.

Gram staining procedure

1. cover smear with crystal violet -

2. gently wash off

3. cover smear with gram iodine

4. gently wash off

5. decolorise with gram decoloriser until obvious reduction in blue colour

6. gently wash off

7. Cover smear with safranin counterstain

8. gently wash off and blot dry

Gram+ or Gram-

Gram+ = blue/violet

Gram- = pink/red

Colony characteritsics of pathogens and commensals of the skin

crowded colonies-smaller due to greater competition

Genetic variation can affect features of a colony like how smooth or rough it is

Pathogenic strains can produce toxins or enzymes to lyse or disrupt host cells (haemolysis- break RBC)

How to describe colony characteristics

-Size (mm)

-Shape

-Elevation (flat,convex,concave)

-Surface appearance (smooth,shiny,rough)

-Type of margin (entire,undulate)

-Colour (white,golden,off-white/cream)

-Opacity

-Presence or absence of changes in surrounding medium

E.coli histology

Staph. aureus histology

Staph. aureus nutrient agar

Golden yellow

Opaque

Convex

Round colonies with sharp,clear edges

Coagulase positive

Catalase positive

Gram +

Staph. aureus blood agar

Staph. epidermidis nutrient agar

White/off-white

Opaque

Convex

Round colonies with sharp, clear edges

Coagulase negative

Catalase positive

Gram +

Staph. epidermidis blood agar

Pseudomonas aeruginosa nutrient agar

Pseudomonas aeruginosa nutrient agar

Strep. pyogenes blood agar top lit

Strep. pyogenes blood agar back lit

Cream/ off white colonies

Raised opaque centres

Large zone of complete haemolysis

Lancefield group A

Ctalase negative

Gram +

Dairy cow with mastitis. Identify the 3 species?

1. E.coli

2. Staph. aureus

3. Staph. epidermidis

One Gram-positive coccus is a round yellow colony, and after staining appears as grape-like clusters, suggesting Staphylococcus aureus. The other Gram-positive coccus is white, and smaller than the yellow colonies, suggesting Staphylococcus epidermidis. The Gramnegative bacillus shows larger cream colonies suggesting E.coli or Salmonella (rather than Pseudomonas which would release a coloured pigment). In this context E.coli is most likely.

Viridans strep.

Small colonies

Grey-green partial haemolysis

Catalse negative

Gram +

Strep. pneumoniae

Small

Water-clear colonies

Often flat or convex

Partial haemolysis

Lancet shaped diplococci

Capsular polysaccharides types with antisera

Catalse negative

Gram +

Enterococcus faecalis

Gram +

Gram-

Gram + rod

Gram + rod

Gram - rod

Anaerobic growth

Some organisms will only grow at temp. below 37 degrees

Some mycobacterium

Issues that can arise when interpreting plate cultures

Haemolysis

Breakdown of erythrocytes due to bacterial proteins being secreted that are pore-forming or virulence associated enzymes that degrade phospholipids…

Incomplete or partial haemolysis

Complete haemolysis

Non-haemolytic