Bacteriology Practical Flashcards
1
Q
Why might staphylococcus lose staining ability?
A
Aging population
Cells degenerate
2
Q
What do bacteria stain in haematoxylin/eosin staining?
A
Nearly all blue, irrespective of gram behaviour
3
Q
What is the analytical profile index?
A
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.
4
Q
Staphylococcus aureus: Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
Round, shiny, golden Sphere Positive Occasionally FAN Catalase positive, coagulase positive, sensitive to antibiotics, oxidase -ve
5
Q
What does staph aureus do pathogenically?
A
Skin infections (boils)
Nosocomial infections e.g. MRSA
Mastitis
Food poisoning from ingested superantigen toxin
6
Q
Staphylococcus epidermidis: Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
COMMENSAL Round, shiny, white Sphere Positive None FAN Catalase positive, coagulase negative
7
Q
How do you differentiate between staphylococcus aureus and staphylococcus epidermidis?
A
S.A = golden colonies, has sometimes got haemolysis, coagulase positive (last one mostly used) S.E = white colonies, no haemolysis, coagulase negative
8
Q
Describe the staphylococcal coagulase test
A
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
9
Q
Which factors can affect colony morphology?
A
Crowded smaller due to competition for nutrients or the formation of inhibitory substances
Genetic variation
10
Q
What are you testing for on a blood agar plate?
A
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
11
Q
Which ‘wound’/skin bacteria are gram + ?
A
S. epidermidis S. Aureus Clostridium perfringens Clostridium tetani Clostridium sporogenes
12
Q
Which wound/skin bacteria are gram - ?
A
Pseudomonas aeruginosa
E coli
13
Q
What does MacConkey medium show?
A
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
14
Q
Name a lactose fermenting bacteria. How do you test for it?
A
E. coli
Grows red/purple on Macconkey plate
15
Q
E. Coli Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
Large, watery margins Fat rod Negative None FAN Grows red on Macconkey agar Grows yellow on CLED Both wound and enteric
16
Q
Where do you label an agar plate?
A
Label base not lid
17
Q
How do you take a throat swab?
A
- 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
18
Q
Enterococcus faecalis Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
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
19
Q
How do you differentiate between E. coli and enterococcus faecalis on a macconkey plate?
A
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 -)
20
Q
Pseudomonas aeruginosa Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
Very large, green Rod -ve None OA Oxidase positive Not generally disease causing but can infect burns, contact lens conjunctivitis
21
Q
Streptococcus pyogenes Colony morphology Organism morphology Gram staining Haemolysis O2 Further tests
A
Round milky Ovoid Positive Complete haemolysis FAN Catalase negative, Lansfield A
22
Q
How do you differentiate staphylococci from enterococci?
A
Catalase test
Staphylococci +
Enterococci -
23
Q
Describe the catalase test
A
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
24
Q
How do you gram stain?
A
- 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
25
Name the families of bacteria we have to know that are gram + cocci
Staphylococcus FAN
| Streptococcus FAN
26
Name the families of bacteria we have to know that are gram - cocci
Neisseria (grow aerobically in 5-10% CO2)
27
Name the families of bacteria we have to know that are gram + rods
Corynebacterium (OA)
| Clostridium (OAN)
28
Name the families of bacteria we have to know that are gram - rods
Escerichia (FAN)
Salmonella (FAN)
Pseudomonas (OA)
Bacteriodes (OAN)
29
How do staphylococci sit together?
Grape-like clusters from broth
Aureus = orange yellow, coagulase +
Epidermidis = off-white, coagulase -
30
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
31
How do enterococci sit together?
Diplococci, chains from broth
| Usually not haemolytic, lancefield D
32
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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
```
33
```
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
```
34
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Clostridium tetani
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
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Swarming
Rod, terminal spore
Positive
None
OAN
Metronidazole sensitive
```
35
What is the test for obligate anaerobes?
Metronidazole sensitive toxicity
| So - Clostridia, bacteriodes
36
Do facultative anaerobes grow better or worse in the presence of O2?
Better
37
Why is metronidazole selective for OAN?
Activation requires reduction of the nitro group
Aerobes and FAN don't have enzymes which can produce a low enough redox potential to do it (PDH and NAD/NADH)
OAN have ferredoxin and other electron transport mechanisms that can
Reduced metronidazole binds to DNA under anaerobic conditions, causes strand breakage and degradation of the molecule. Also inhibits a DNA repair enzyme.
38
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Bacteriodes fragilis
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Enteric (part of normal flora LI, can cause wound infections/septicaemia after surgery)
Small, shiny, foul
Rod
Negative
None
OAN
Metronidazole sensitive
```
39
How do you stain for bacterial spores?
Hot malachite green stain
40
Describe the hot malachite green stain
1. Prepare normal smears
2. Cover with malachite green and heat gently on the hotplate for at least 5 mins, not letting the stain dry
3. Wash
4. Counterstain with safranin for 5 mins
5. Wash, blot, examine under oil immersion
6. Spores stain green, vegetative cells stain pink/red
41
What does safranin stain?
Stains all cell nuclei red
42
Where are the spores located in the 3 clostridia species?
C perfringens = mid spore
C sporogenes = sub-terminal spore
C tetani = terminal spore
43
What is the Nagler Reaction?
Test for clostridium perfringens alpha toxin
Agar containing egg yolk (source of lecithin)
C perfringens produces an opaque zone representing insoluble diglyceride around the colonies
Anti-toxin abolishes the white zone
44
What is the clostridium perfringens alpha toxin?
A lecithinase that hydrolyses the phospholipid lecithin (in cell membranes) to a diglyceride and phosphorylcholine
45
What effect does clostridium perfringens have on skeletal muscle?
Gas gangrene
Necrosis - lose cell definition, lose nuclei, cells very eosinophilic
H2 produced - when you press on it you hear cracking
46
What does ATCC stand for?
American type culture collection
47
How do you account for the inherent differences between the antibiotic sensitivities of different strains of bacteria?
Different selective pressures
| Random mutations
48
Which bacteria produce beta lactamases?
Penicillin resistant S Aureus
N gonorrhoeae
Ampicillin-resistant haemophilus influenzae
49
How do you test for the presence of beta lactamases?
They convert penicillin into penicilloic acid
pH change
Bromothymol blue changes to yellow below pH 6 from blue above 7.6 in the presence of the acid (apply a heavy loop of bacteria)
50
What is gas-liquid chromatography used for?
Obligate anaerobic infections
• Used routinely to
1. Identify pure cultures grown in vitro: different genera and species vary in the range of volatile fatty acids produced, and the patterns of metabolic end products are stable and reproducible, so of value in identification
2. Detection of anaerobes grown in clinical material: detection of volatile fatty acids in pus can detect anaerobic infection. Pus from aerobes or FAN contains no detectable volatile fatty acids, or acetic and propionic acids only.
51
Where are clostridia found in nature?
Saprophytes in soil
| Alimentary tracts
52
When are clostridia infections most dangerous?
Blood supply to wound is poor
| Wound infected by FAN like E coli, staphylococci and streptococci which further lower the redox infection
53
Treatment for gas gangrene?
radical surgery, antitoxin therapy, antibiotics, nursing in hyperbaric oxygen-rich atmosphere
54
What are the toxins of type A c perfringens?
1. Alpha toxin lecithinase/PLC - Ca2+ Mg2+ dependent. Lethal, necrotising, haemolytic
2. Collagenase, hyaluronidase, DNAase
3. Theta-toxin: haemolysis on horse blood agar. Also antigenically cross reacts with streptolysin O and tetanolysin
55
How do you grow an anaerobic culture?
Airtight jar containing a catalyst
| + a Gaspak: disposable H2 generator, allows O2 to be combined with H2, CO2 alos formed
56
```
Streptococcus pneumoniae
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Bacterial pneumonia, ear infections, meningitis. May also be a normal component of the flora of the nasopharynx.
Draughtmans shape
Ovoid diplococcus
Positive
Incomplete
FAN
Catalase -ve, encapsulation
```
57
```
Streptococcus Viridans
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Round, milky
Ovoid
Positive
Incomplete
FAN
Catalase negative
```
58
```
Corynebacterium diphtheria
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Pale grey
Rod, Chinese letters
Positive
None
OA
Catalase positive, polyphosphate granules (Albert's stain), ELEK test for toxin
```
59
```
Neisseria pharynges
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Small, shiny, dove grey
Diplococci
-ve
None
OA (grow ideally in 5-10% CO2)
Oxidase positive
```
60
Which respiratory bacteria show complete haemolysis and which show incomplete haemolysis?
Complete: Strep pyogenes, staph aureus often
Incomplete: Strep pneumonia, strep viridans
None: Staph epidermidis, corynebacterium diphtheria, Neisseria pharynges (candida albincans)
61
Which is the one respiratory pathogen which is gram -ve?
Neisseria pharynges
62
How do you differentiate between streptococci?
Lansfield staining
63
Describe the Lansfield test
* Immunological test
* Most species of Streptococci have characteristic carbohydrate antigens in their cell walls
* Extract antigens in their soluble form
* Mix with latex particle pre-coated with Lansfield group-specific antibodies
* Positive result seen when latex particles agglutinate in the presence of cognate antigen
* Using these, can divide into 3 groups A B G
64
Give an example of a Lansfield group A and C
Strep pyogenes = A
| Strep equi = C
65
What can strep pyogenes lead to?
Rheumatic fever
| Acute post-streptococcal glomerulonephritis
66
Describe the titration of anti-streptolysin O by toxin neutralisation
1. Serum heated to inactivate complement
2. Dilute (starting 1:50)
3. Titrate against streptolysin O prepared from a filtrate of a broth culture of strep pyogenes + reducing agent to protect it from inactivation by oxidation
4. Incubate 15 min at 37 degrees
5. Add horse RBC suspension
6. Incubate for 1h
7. Read end point: last one without haemolysis
67
What controls do you use for an anti streptomycin O titration?
Controls: check it’s the extract lysing and not the serum
1. Extract and RBC: checking extract does haemolyse
2. Serum and RBC: checking serum doesn’t spontaneously haemolyse
68
What does the Albert's test test for?
Corynebacterium diphtheria
| Stains polyphosphate granules
69
What are 'Chinese characters'?
Corynebacterium diphtheria contain polyphosphate granules that form clumps that look like kanji when stained with Albert's stain
70
What are the tests for Corynebacterium diphtheria?
Catalase positive
Albert's stain polyphosphate granules
ELEK test for toxin
71
What does the ELEK plate test for?
Diphtheria toxin
| So differentiates between toxinogenic and non-toxinogenic strains
72
Describe the ELEK test
* Grow C diptheriae in thick streaks at right angles to a strip of filter paper that has been soaked in antitoxin
* Lines of ppt form after several days at the sites where toxin and antitoxin meet
73
What carries the gene for the diphtheria toxin?
A beta bacteriophage
74
What is the oxidase test?
Test for Neisseria and pseudomonas (OA)
Transfer colony to oxidase detection strip using a loop. Spread and observe for up to 5 seconds. A deep blue violet colour indicates a positive reaction (i.e. has cytochrome oxidase)
75
```
Candida albicans
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
YEAST, EUKARYOTE
Small white round
Dimorphic: most commonly budding round or oval yeast, but filamentous hyphae present in invasive lesions
Positive, stain dark blue/black
None
Grow on mac, white
Superficial oral, genitourinary infections common
76
```
Salmonella spp not typhi
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Gastroenteritis, enteric fever, septicaemia
Large watery margins
Rod with peritichous flagella
Gram -ve
None
FAN
Group by O, H serotypes
White on mac
CLED pale green/blue
```
77
```
Neisseria gonorrhoeae
Colony morphology
Organism morphology
Gram staining
Haemolysis
O2
Further tests
```
```
Round, white
Diplococci but can be tetrads
Negative
No
Oxidase positive
```
78
Which tests use bromothymol blue?
Test for beta lactamases
| CLED
79
What does CLED stand for?
Cysteine lactose electrolyte deficient agar
80
What do you test CLED with?
Urine samples
81
Describe CLED
* Differentiates lactose and non-lactose fermenters
* Indicator bromothymol blue (same as test for beta lactamases)
* Yellow at acid pH
82
Which bacteria are yellow on CLED?
E coli
| Enterococcus faecalis
83
Which bacteria are pale blue/green on CLED?
Salmonella
84
What is the point of CLED being electrolyte deficient?
Prevents proteus mirabilis swarming
85
What antigens are used in serological identification of salmonella?
H flagellar
O somatic
Vi
86
Describe serological testing of salmonella
* Mix suspension on a glass tile with the antisera specific for particular O and H antigen serotypes.
* Bacteria clumping = positive
