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Labs Flashcards

Gram Positive Cocci

1
Q

Gram Positive Cocci

A

Staph
Strep

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2
Q

GPC Grape-like cluster

A

Staph

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3
Q

GPC chains + clusters

A

Strep

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4
Q

Staph culture conditions

A

Aerobic 37C Blood Agar

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5
Q

Strep culture conditions

A

Aerobic 37C CO2 Blood Agar

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6
Q

Staph colony morphology

A

2-3mm round raised white/cream

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7
Q

Colony morphology:
Blood Agar
2-3mm, round, raised, white/cream.
No haem

A

?Staph

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8
Q

Presumptive Staph tests

A
  1. Tube coagulase test
  2. Staphaurex Plus test
  3. DNase test
  4. S/D - Mannitol Salt agar/ CHROMID agar test.
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9
Q

Staph Aureus infections

A

Superative infections:
1. Infection of hair follicles - folliculitis, furuncles, carbuncles.
2. Impetigo (pustular, bullous)
3. Cellulitis
4. Mastitis
5. Surgical wounds
6. Abscesses
Systemic infections:
1. Meningitis
2. Pneumonia
3.Endocarditis
4.Pyelonephritis
5. Biofilm on medical devices
Toxin-mediated
1. TSS
2. Necrotizing pneumonia (PVL)
3. SSSS (exfoliative toxins)
4. Food poisoning (enterotoxins)

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10
Q

Staph Aureus food poisoning

A

Enterotoxin
Acute onset 2-6hrs
Severe nausea + vomiting + abdominal pain + diarrhea
Self-limiting 8-24hrs
If disseminates to blood can cause TSS

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11
Q

Staph aureus food poisoning sources

A

Food prepared by handlers + no additional cooking
-salads, ham, egg, tuna, chicken, potato,
- dairy products + milk
- Bakery (cream pastries)
- sandwiches

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12
Q

Staph epidermidis infections

A
  1. Device associated infections - IV, feeding lines, catheters, implanted prosthetic devices.
  2. HCAI bloodstream infection.
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13
Q

Staph saprophyticus infection

A
  1. UTIs in sexually active young females
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14
Q

Staph specimens

A

Skin swab, pus specimen, wound swab, blood, sputum, urine

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15
Q

Additional confirmatory tests Staph Aureus

A
  1. VITEK GP card = extra biochemical tests
  2. MALDI TOF = protein profile
  3. PCR = Xpert MRSA/ SA assay - target Protein A (SA) or mecA (MRSA) DNA sequences.
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16
Q

Staph aureus - what to do if clinically significant

A

AST + Treatment
No resistance = methicillin
MRSA = vancomycin

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17
Q

Staph aureus superficial infection treatment

A

Topical = Mupirocin
Oral = Augmentin

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18
Q

S epidermidis infection treatment

A

Vancomycin
If MRSA = oxacillin + rifampcin
Skin infection = Fucidic acid

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19
Q

S. Saprophytic infection treatment

A

UTI - Nitrofurantoin

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20
Q

Streptococcus pneumonia infections

A

Non-invasive
1. Acute bacterial pneumonia = LRT (older, can spread to BS - sepsis)
2. Otitis Media
Invasive
3. BSI - Sepsis
4. Meningitis

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21
Q

Strep specimens

A

Sputum, pleural/lung fluid, pus, aspirates.
CSF, blood taken straight for molecular detection.

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22
Q

Strep pneumonia colonies types

A

Alpha haemolysis
Draughtsman colonies (flat w/ depressed centre)
Mucoid colonies

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23
Q

Strep pneumonia Basic Characterisation

A

Gram positive diplococci (lancet-shaped) - chains + clusters
Catalase negative !!
Oxidase negative
KOh negative

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24
Q

Presumptive a-haem Strep tests

A
  1. Optochin susceptibility test
    = S. Pneumonia is susceptible = clear zone
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25
Strep pneumonia additional confirmatory tests
1. Vitek/ MALDI TOF identification - MALDI not very good at distinguishing a-haem strep! 2. CSF sent to IMSRL - Temple Street perform real time PCR on capsule to serogroup.
26
Strep pneumonia- what to do if clinically significant
1. AST 2. Penicillin , Erythromycin, Cefotaxime
27
Staph tube coagulase test procedure
1. emulsify 1 colony of test organism in 1ml rabbit plasma. 2. Incubate at 37C overnight. Pos = visible clot formation or lose web of fibrin. Neg = No clot.
28
Staphaurex Plus kit procedure
1. Bring latex to room temp + mix by vigorous shaking. 2. Dispense one drop of test latex onto one circle and one drop of control on the other. 3. Using loop, pick up and smear equivalent of 5 colonies onto circle + mix. 4. Spread to cover circle. 5. Rock card for 20 s. 6. Observe for agglutination. Agglutination within 20s = S. Aureus No agglutination = Coagulase-neg. Staph.
29
Staph DNase test procedure
1. Divide DNA agar plate into 3 2. Spot inoculate test on 1 half (10mm) and controls (pos = S.Aureus, neg = S.epidermidis) on other half. 3. Incubate at 37C overnight. 4. Following incubation, in fume hood flood plate with 1M HCl and allow acid to permeate for 10 mins and carefully pour off excess into discard jar. Pos = distinct clear zones around inoculum = Staph Aureus Neg = S. epidermidis.
30
Staph Mannitol Salt agar + SAIDE agar test procedure + results
1. Using streak-inoculation, sub-culture test organisms + controls (SA, SE) isolates on MS agar and SAIDE agar 2. Incubate overnight at 37C. S.Aureus = growth + fermentation on MS (Pink -> Yellow) + growth on SAIDE. S. epidermidis = growth + no fermentation on MSA (Pink) + no growth on SAIDE.
31
Strep Pneumonia Optochin Susceptibility Test procedure
1. Divide BA plate into 3 sections. 2. Prepare suspension in reduced volume of sterile water. 3. Lawn one half of the agar with suspension of test organism 4. Lawn other half with Pos control (S.Pneumonia) + Neg control (S. Viridans). 5. Place optochin disk in centre of each inoculum. 6. Incubate BA plate overnight at 37C. Positive = zone of inhibition >5mm = S.pneumoniae Negative = no inhibition = Strep. viridans.
32
Strep Viridans spp (2) + infection
Strep oralis/mitis -> Streptococcal Endocarditis - infection of inner most layers of the heart. Strep mutans -> biofilm in mouth/teeth - plaque formation + tooth decay.
33
S. viridans basic characteristics
Gram positive cocci in chains Catalase negative Oxidase negative Facultative anaerobe. Fastidious
34
Beta haemolytic strep colonies
Beta haemolysis Pinpoint colonies Colourless.
35
Presumptive B-haemolytic Strep tests
1. Lancefield Grouping 2. Bacitracin Susceptibility Testing 3. MacConkey Agar
36
Strep. Lancefield grouping test procedure
Use Oxoid Streptococcal Grouping kit Controls = Pos. S.pyogenes, Neg. Enterococcus. 1. Dispense 0.4ml extraction enzyme into labelled test tube + add 2-5 test colonies. 2. Incubate for 10 mins at 37C in water bath. 3. After 5 mins incubation remove tube and shake vigourly for 2-3seconds, continue incubation. 4. Remove and allow to cool to room temp. Agglutination test: 1. Bring latex reagents to room temp by warming in hands + mix by shaking. 2. Dispense 1 drop of each latex reagent (A,B,C, D, F, G) in the circular rings on card. 3. Using pipette add 1 drop of extract to each of the 6 rings. 4. Mix using separate stick for each ring. 5. Gently rock card - agglutination should occur within 30s. Group A = Strep pyogenes Group B = Strep agalactiae Group D = Enterococcus
37
BH Strep Bacitractin susceptibility test procedure
Used for Group A (S.pyogenes) identification 1. Divide blood agar into 3 2. Prepare suspension using reduced water volume. 3. Lawn one half with test organism + other half with controls (pos= S.pyogenes, neg= enterococcus). Leave margins. 4, Place bacitracin disk in centre of each inoculum. 5. Incubate BA plate overnight 37C. Pos = zone of inhibition >5mm = S. pyogenes (group A).
38
BH Strep MacConkey Agar Test procedure
Lancefield groups A, C, G will NOT grow on media containing bile. Group D grows on media with bile. 1. Split agar into 3. 2. Light zig-zag inoculum of test organism + PC (Enterococcus) + NC (S.pyogenes) on MacConkey agar. 3. Incubate plates at 37C overnight. Positive = Group D (enterococcus) will grow - magenta colonies.
39
Strep pyogenes infection
Non-invasive skin infection 1. Pharyngitis (URT) 2. Scarlet fever 3. Skin infections - impetigo, cellulitis, erysipelsia Invasive skin infection 4. Necrotising Fascitis Toxin mediated 5. TSS 2. Scarlet fever Non-suppurative sequelae (1-5wks post infection) 6. Rheumatic fever 7. Glomerulus Nephritis
40
Presumptive non-haem Enterococci tests
1. Lancefield grouping 2. MacConkey agar 3. Bile Aesculin test
41
Non-haem strep Bile Aesculin test procedure
Identify Group D Strep 1. Split agar into 3 2. Spot inoculate test organism onto plate as 10mm circle 3. Inoculate PC (Enterococcus) + NC (S.pyogenes) onto other half. 4. Incubate plate at 45C overnight + record blacking of medium. Growth + black = enterococcus
42
Beta haem Strep treatment
Penicillin Clarithromycin (allergy) Necrotizing fascitis = surgery + clindamycin
43
Enterococci infections
Normal commensal of GI tract Most infections are endogenous Often HAI 1. UTI 2. Intra abdominal abscess 3. Endocarditis 4. BSI
44
Enterococci specimens
Urine samples - UTI Blood cultures -BSI Pus
45
Enterococci - what if it is clinically significant
1. AST 2. Treatment - Ampicillin+ Gentamycin - Vancomycin (many show resistance).
46
Alpha haem Strep test materials
1. Blood Agar (1) 2. Bijou (3) 3. Optochin disks (3) Controls = S.pneumonia, S.viridans
47
Beta haem Strep tests materials
1. Oxoid Streptococcal grouping kit 2. Wooden picks Controls = S.pyogenes, Enterococcus 3. Blood Agar (1) 4. Bijou (3) 5. Bacitracin disks (3) 6. MacConkey agar
48
Non haem Strep test materials
1. Bile Aesculin agar Controls = Enterococcus, S.pyogenes.
49
Gram Negative Dipplococci
Neisseria spp. Moraxella spp.
50
Grows on Chocolate + Blood agar 5-10% CO2, 37C
Neisseria, Moraxella, Haemophilus
51
Neisseria culture conditions
5-10% CO2, 37C, chocolate agar (maybe blood agar too)
52
Neisseria colonies on chocolate agar
1-2mm, grey, round, smooth, wet, no haem
53
Moraxella spp colony morphology
1-3mm, grey, round, smooth, dry, no haem
54
Neisseria spp. basic characteristics
Gram negative dipplococci (coffee bean shaped) KOh positive Oxidase positive Catalase positive
55
Moraxella spp basic characteristics
Gram negative diplococci (coffee bean - coccobacilli in pairs) KOH positive Oxidase positive Catalase positive
56
Bacteria that do not grow on blood agar
Haemophilus spp.
57
Chocolate agar 1-2mm convex, grey-white colonies
Haemophilus
58
Presumptive Haemophilus tests
1. X and V Growth factors
59
Haemophilus spp. basic characteristics
Gram negative coccobacilli, KOH positive Oxidase Positive Catalase positive (h. influenza) / negative (H. parainfluenza)
60
Oxidase Positive bacteria
Neisseria, Moraxella, Haemophilus,
61
Catalase negative bacteria
Streptococcus, haemophilus parainfluenza Clostridioides
62
Gram positive bacilli Aerobic
Corynebacteria, Bacillus
63
Corynebacteria culture conditions
Aerobic 37C Blood agar
64
Bacillius culture conditions
Aerobic 37C Blood agar
65
Corynebacteria colony morphology on BA
1-2mm round, dry, grey/cream weak b-haemolysis
66
Bacillus colony morphology on BA
2-5mm, dry, grey-yellow, irregular edge, b-haemolysis
67
Corynebacteria basic characteristics
Gram positive bacilli Chinese-lettering appearance (clusters) KOH negative Oxidase negative Catalase positive
68
Presumptive corynebacteria spp. tests
1. Trehalose fermentation 2. Urease Production 3. Tinsdale medium
69
Presumptive Bacillus spp. tests
1. Selective agar = PEMBA 2. Motility test (Hanging drop test)
70
Anaerobic bacteria
Clostridioides Bacteroides
71
Anaerobic Gram positive bacillus
Clostridioides
72
Anaerobic Gram negative bacillus
Bacteroides
73
Preliminary Haemophilus app test
X and V Growth Factors 1. Prepare heavy inoculum (1-2 colonies in reduced water) 2. Lawn DST agar evenly 3. Allow suspension to dry 4. Use sterile forceps, place X, Y, XY disks in petri plate (3.5cm away) 5. Incubate at 5% CO2 overnight at 35-37C 6. Observe growth of bacteria in vicinity of disks. Visible colonies around XV only = H. influenza Visible colonies around XV + V = H. parainfluenza.
74
75
Bacillus spp. Basic characteristics
Gram positive bacillus Large rods Endospores KOH neg Catalase postive Oxidase negative.
76
Gram positive bacillus Large rods Endospores KOH neg Catalase postive Oxidase negative.
Bacillus spp.
77
Gram positive bacilli Chinese-lettering appearance (clusters) KOH negative Oxidase negative Catalase positive
Corynebacterium spp.
78
Corynebacterium spp. Trehalose fermentation + Urease Production test procedure
1. Label each tube appropriately + loosen the caps. 2. Label blood agar plate = purity plate check. 3. Inoculate trehalose peptone water sugar - tilt liquid media to the right an gently 'rub' inoculate loop on the left side of the tube just below meniscus. Turn upright + gently shake. 4. Immediately after inoculation of trehalose, without flaming, inoculate urea medium by zig-zagging over surface of the agar and 'stabbing' the slop with the loop. 5. Immediately after inoculating the urea slop, without flaming, inoculate the blood agar using quadrant streak method. 6. Incubate the inoculated trehalose, urea agar slope and blood agar purity plate at 37C overnight.
79
Corynebacterium Trehalose + Urease biochemical test results
Trehalose Positive = pink -> yellow + presence of bubble (indicates gas production). Negative = remains pink. Urease: Positive = Yellow -> Pink Negative = remains yellow.
80
Corynebacterium Tinsdale medium test procedure
1. Streak inoculate Tinsdale selective media. 2. Let loop dig into agar in places with Tinsdale medium. 3. Incubate plate at 37C. 4. Read at 24hrs + 48hrs.
81
Corynebacterium Tinsdale medium test - colonial characteristics
C. diphtheriae - gravis = small, shiny black. C. diphtheriae - mitis/ulcerans = convex colonies with dark brown halos after 24hrs. C. dipththeriae - intermedius = Pale brown colonies with form halos after 36hrs incubation. Diphtheroids = dark brown colonies without halos - no discolouration of the medium.
82
Tinsdale medium - C. diphtheriae - gravis
Small shiny black colonies
83
Tinsdale medium = C- diptheriae - mitis C. ulcerans
Convex colonies with dark brown halos after 24hrs incubation.
84
Tinsdale medium = C. diphtheriae biotype intermedius
Pale brown colonies which form halos after 36hrs incubation.
85
Tinsdale medium = Diphtheroids
Dark brown colonies without halos - minute colonies showing no discolouration of the medium
86
Bacillus spp. PEMBA test procedure
1. Streak inoculate test organism onto B.cereus selective media (PEMBA plate - lime green). 2. Incubate plate at 37C. Read at 24hrs + 48 hrs.
87
B. cereus PEMBA plate test results
B.cereus positive = crenated (rounded scallop edges), 5mm, distinctive turquoise/ peacock blue colour surrounded by egg yolk precipitate of same colour.
88
Corynebacteria diphteria test results (1. Chinese lettering, 2. fermentation of trehalose, 3. urease production, 4. growth on tinsdale medium)
1. Chinese lettering positive 2. No fermentation of trehalose (remains pink) 3. No urease production (remains yellow). 4. Growth on tindsale medium positive.
89
Corynebacteria ulcerans test results (1. Chinese lettering, 2. fermentation of trehalose, 3. urease production, 4. growth on tinsdale medium)
1. Chinese lettering positive 2. Fermentation of trehalose (turns yellow) 3. Urease production (turns pink). 4. Growth on tindsale medium positive.
90
Corynebacteria pseudotuberculosis test results (1. Chinese lettering, 2. fermentation of trehalose, 3. urease production, 4. growth on tinsdale medium)
1. Chinese lettering positive 2. No fermentation of trehalose (remains pink) 3. Urease production (turns pink). 4. Growth on tindsale medium positive.
91
Diphtheroids test results (1. Chinese lettering, 2. fermentation of trehalose, 3. urease production, 4. growth on tinsdale medium)
1. Chinese lettering positive 2. No fermentation of trehalose (remains pink) 3. Urease production (turns pink). 4. Growth on tindsale medium positive.
92
Bacillus cereus test results: 1. Haemolysis 2. Egg yolk reaction 3. Fermentation 4. Motility
1. Haemolysis positive 2. Egg yolk precipitation positive (lecithinase positive) 3. No fermentation of mannitol ( blue colonies) 4. Motile *Same results as B. thuringenesis
93
Bacillus thuringenesis test results: 1. Haemolysis 2. Egg yolk reaction 3. Fermentation 4. Motility
1. Haemolysis positive 2. Egg yolk precipitation positive (lecithinase positive) 3. No fermentation of mannitol ( blue colonies) 4. Motile *Same as B.cereus!
94
Bacillus anthracis test results: 1. Haemolysis 2. Egg yolk reaction 3. Fermentation 4. Motility
1. Haemolysis negative 2. Egg yolk precipitation is variable + narrow zone (lecithinase positive) 3. No fermentation of mannitol ( blue colonies) 4. Not motile
95
Bacillus subtilis test results: 1. Haemolysis 2. Egg yolk reaction 3. Fermentation 4. Motility
1. Haemolysis negative 2. No egg yolk precipitation (lecithinase negative) 3. Fermentation of mannitol (white colonies) 4. Motile
96
Corynebacterium Trehalose + Urease test controls
Positive control = C.ulcerans Negative control = E.coli
97
Gram positive bacillus
Corynebacterium (aerobic) Bacillus (aerobic) Clostridioides (anaerobic)
98

Medical Microbiology Labs (14 decks)

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