Clostridioides spp

Question 1

Culture conditions

Answer 1

Anaerobic 37C blood agar

Question 2

Anaerobic bacteria

Answer 2

Clostridioides
Bacteroides

Question 3

Anaerobic Gram positive bacillus

Answer 3

Clostridioides

Question 4

Colonial morphology

Answer 4

Large convex irregular edge double B-haemolysis.

Question 5

Gram stain

Answer 5

Gram positive bacilli
Single endospores

C. perfringens = short fat rods + central spore
C. tetani = drumstick appearance + terminal spore
C. difficile = large spores

Question 6

Endospores location Clostridium spp.

Answer 6

C. perfringens = central spore
C. tetani = terminal spores

Question 7

Biochemical tests

Answer 7

KOH = negative
Catalase = negative
Oxidase = negative

Question 8

Presumptive clostridium spp tests

Answer 8

1. Anaerobic growth tests
2. Metronidazole susceptibility test
3. Lactose gelatin medium
4. Egg yolk agar = Nagler reaction

Question 9

Anaerobic growth test + metronidazole susceptibility test procedure

Answer 9

1. Lawn inoculate blood agar purity plate with test organism + PC ( P. aeruginosa) + NC (C. perfringens) and incubate AEROBICALLY overnight at 37C
2. Lawn inoculate blood agar purity plate with test + controls (PC = C. perfringens, NC = P. aeruginosa).
3. Place metronidazole disc on secondary inoculum of test organism.
4. Incubate ANAEROBICALLY for 48hrs using anaerobic jar and AnaeroGen sachet.

Question 10

Lactose Gelatin media test procedure

Answer 10

1. Using straight wire loop stab - inoculate selected colony into tube
2. Inoculate control strains C.perfringens, C. sporogenes, E.coli
3. Incubate 35C anaerobically for 24-48hrs.
4. Following incubation examine for lactose fermentation
5. Refrigerate tubes until well chilled (1hr) and examine for gelatin liquification.

Question 11

Lactose Gelatin medium test results

Answer 11

Lactose fermentation= presence of bubble + colour change red to yellow
Gelatinase positive = gel is now liquid.
C. perfringens = yellow + liquid (= ferments lactose + gelatinase positive).
C. sporogenes = red + liquid
C. difficile = yellow + liquid
C. tetani = red + gel
C. botulinum = red + liquid.

Question 12

Nagler reaction - egg yolk agar test procedure

Answer 12

1. Divide egg yolk into 2 halves
2. Label one with Cperfringens type A antitoxin A.
3. Wear disposable gloves + safety glasses and swab half plate with antitoxin A
4. Inoculate test + control (C.perfringens) onto half without antitoxin and move across to antitoxin side.
5. Incubate anaerobically at 37C for 48hrs.

Question 13

Nagler reaction principle

Answer 13

Detects lecithinase (phospholipiase) activity + lipase activity
Lecithinase positive -> opaque, diffuse zone around colonies.
Disappearance of opacity on antitoxin hald of plate (= neutralization of lecithinase)
Lipase positive -> iridescent sheen.

Question 14

Nagler reaction test results

Answer 14

C. perfringens = opaque diffuse zone + no sheen
C. sporogenes = no zones + iridescent sheen
C. difficile = no zones + no sheen
C. tetani = no zones + no sheen
C. botulinum = variable zones + iridescent sheen

Question 15

C perfringens diseases

Answer 15

Gas gangrene
Myonecrosis
Food poisoning - diarrhoea (6-18hrs after food consumption + resolves within 1-2 days)

Question 16

C. difficile infection + treatment

Answer 16

Antibiotic associated diarrhoea
Pseudomembrane colitis

Cephalosporins, clindamycin, fluoroquinlones

Question 17

C. botulinum disease

Answer 17

Botulism = flaccid paralysis

Question 18

C. tetani disease

Answer 18

Tetanus = severe prolonged muscle spasms

Question 19

C. perfringens Food Poisoning + Gas gangrene/myonecrosis treatment

Answer 19

Rehydration
IV fluid

Penicillin G

Question 20

Specimens

Answer 20

Would/soft tissue = pus , tissue sample, muscle biopsy
Foodborne infections = faeces, food, gastric contents
Pseudomembrane colitis = faecal specimen.