Bacteriology Practical Flashcards
Why might staphylococcus lose staining ability?
Aging population
Cells degenerate
What do bacteria stain in haematoxylin/eosin staining?
Nearly all blue, irrespective of gram behaviour
What is the analytical profile index?
Metabolic activity of different bacterial species has different effects of different chemical substrates. Can detect effects after incubation overnight by colour changes that either happen spontaneously or after the addition of other reagents.
Staphylococcus aureus: Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Round, shiny, golden Sphere Positive Occasionally FAN Catalase positive, coagulase positive, sensitive to antibiotics, oxidase -ve
What does staph aureus do pathogenically?
Skin infections (boils)
Nosocomial infections e.g. MRSA
Mastitis
Food poisoning from ingested superantigen toxin
Staphylococcus epidermidis: Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
COMMENSAL Round, shiny, white Sphere Positive None FAN Catalase positive, coagulase negative
How do you differentiate between staphylococcus aureus and staphylococcus epidermidis?
S.A = golden colonies, has sometimes got haemolysis, coagulase positive (last one mostly used) S.E = white colonies, no haemolysis, coagulase negative
Describe the staphylococcal coagulase test
Latex bead agglutination
Pipette plasma and broth culture into tube. Mix well, incubate at 37 degrees, 1.5h, remove tubes, hold to light/tilt/rotate/gently shake
+ = clearly visible veil of polymerised fibrin
Which factors can affect colony morphology?
Crowded smaller due to competition for nutrients or the formation of inhibitory substances
Genetic variation
What are you testing for on a blood agar plate?
Pathogenic strains of many bacteria produce toxins or enzymes that lyse RBCs
Put a light source behind the base of the plate and look at the agar surface of the plate
Which ‘wound’/skin bacteria are gram + ?
S. epidermidis S. Aureus Clostridium perfringens Clostridium tetani Clostridium sporogenes
Which wound/skin bacteria are gram - ?
Pseudomonas aeruginosa
E coli
What does MacConkey medium show?
Contains bile salts that inhibit the growth of non-enteric organisms so testing for gut organisms
Contains lactose and a pH indicator (neutral red)
Red/purple at low pH, colourless at neutral/high pH
Lactose fermenting bacteria grow as red/purple colonies (produce acid from lactose fermentation that locally drops pH), non-fermenting grow as pale
Name a lactose fermenting bacteria. How do you test for it?
E. coli
Grows red/purple on Macconkey plate
E. Coli Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Large, watery margins Fat rod Negative None FAN Grows red on Macconkey agar Grows yellow on CLED Both wound and enteric
Where do you label an agar plate?
Label base not lid
How do you take a throat swab?
- Label
- Swab tonsils
- Lift plate from lid and inoculate a small area A with material from swab
- Using a sterile plastic loop spread a portion of the inoculum from A to B
- Repeat to C with a new loop
- Make a single wavy line across the remaining surface with D
- Replace dish in lid
Enterococcus faecalis Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Round milky chains Ovoid Positive None FAN Catalase negative, grows on Macconkey red colony Yellow on CLED Commensal GI, also faecal contamination of superficial wounds
How do you differentiate between E. coli and enterococcus faecalis on a macconkey plate?
Both red
So look at colony morphology (E faecalis round milky chains, E. coli large watery margins)
Organism morphology (EF ovoid, E coli fat rod)
Gram stain (EF +, EC -)
Pseudomonas aeruginosa Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Very large, green Rod -ve None OA Oxidase positive Not generally disease causing but can infect burns, contact lens conjunctivitis
Streptococcus pyogenes Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Round milky Ovoid Positive Complete haemolysis FAN Catalase negative, Lansfield A
How do you differentiate staphylococci from enterococci?
Catalase test
Staphylococci +
Enterococci -
Describe the catalase test
Touch colony with one end of a capillary tube, without blocking it
Dip the other end in H2O2
Tilt tube so peroxide runs into the bacterial sample
+ve = oxygen evolved within the capillary
How do you gram stain?
- Transfer a minimum loop of distilled water to a slide
- Use the edge of another loop to pick up part of a colony
- Spread the organisms over a small area of the slide, making a smear of the bacteria in the distilled water
- Dry on a hotplate
- Leave to cool
- Cover with crystal violet for 1 min
- Rinse with tap water
- Cover with Gram’s iodine for 1 min
- Rinse with tap water
- Decolorise with decoloriser until obvious blue colour is reduced or doesn’t wash out easily (5-10s)
- Rinse with tap water
- Safranin counterstain 30-60s
- Blot and dry
Name the families of bacteria we have to know that are gram + cocci
Staphylococcus FAN
Streptococcus FAN
Name the families of bacteria we have to know that are gram - cocci
Neisseria (grow aerobically in 5-10% CO2)
Name the families of bacteria we have to know that are gram + rods
Corynebacterium (OA)
Clostridium (OAN)
Name the families of bacteria we have to know that are gram - rods
Escerichia (FAN)
Salmonella (FAN)
Pseudomonas (OA)
Bacteriodes (OAN)
How do staphylococci sit together?
Grape-like clusters from broth
Aureus = orange yellow, coagulase +
Epidermidis = off-white, coagulase -
How do streptococci sit together?
Diplococci
Chains from broth
Strep viridans = haemolytic, tiny colonies 0.5mm
Strep pneumonia = haemolytic, 2mm colonies water-clear colonies, flat topped or sunken, capsular polysaccharides typed with antisera
Strep pyogenes = haemolytic, lancefield test for cell-wall polysaccharide
How do enterococci sit together?
Diplococci, chains from broth
Usually not haemolytic, lancefield D
Clostridium perfringens Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Gives gas gangrene, food poisoning Large flat volcano Rod, mid spore Positive Complete and incomplete - two zones of lysis due to a and t toxins OAN Metronidazole sensitive Phospholipase C positive Nagler reaction Separated into types A to E depending on toxin spectra A = gas gangrene B to E = opportunist pathogens C = rare cause of lethal necrotic enteritis in man
Clostridium sporogenes Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Small, medusa head, foul Rod, sub-terminal spore \+ Haemolytic (??) OAN Metronidazole sensitive Produce volatile fatty acids + alcohols, detect by gas/liquid chromatography of culture filtrates Markedly proteolytic so smell of H2S from putrefying flesh Non toxinogenic
Clostridium tetani Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
Swarming Rod, terminal spore Positive None OAN Metronidazole sensitive