Practical 7: Investigation of Gram-positive Anaerobic Bacilli Flashcards
What are the gram positive anaerobic bacilli?
(2)
Clostridium
Bifdobacteria
What tests do you use to investigate gram-positive anaerobic bacilli
Anaerobic growth and metronidazole susceptibility
Lactose Gelatin Medium
Growth on Egg yolk agar -> the Naglar reaction
What are the clinically significant species of Clostridium
C. botulinum
C. difficile
C. perfringens
C. tetani
Where is C. botulinum found and what infections does it cause?
(2)
Its an organism that produces a toxin in food/wound
It causes botulism
Where is C. difficile found and what infections does it cause?
Often overgrows commensals in the gut during antibiotic therapy and can cause pseudomembranous colitis
Where is C. perfringens found and what infections does it cause?
It causes a wide range of symptoms from food poisoning to gas gangrene
Where is C. tetani found and what infections does it cause?
Causes tetanus
What is the principle behind using lactose gelatin medium
Some bacteria demonstrate proteolytic activity that results in the liquidification of gelatin
Lactose is added to the medium to investigate fermentation
Used to identify C. perfringens
How do you carry out a lactose gelatin medium test?
(4)
Ask for a straight wire loop and a lactose gelatin tube
Inoculate test and controls (+ C. perfringens and -C sporogenes and E.Coli)
Incubate at 35 degrees
Refrigerate until chilled and examine for gelatin liquidisation and lactose fermentation
What result will C. perfringens give in a lactose gelatin medium?
Yellow liquid
Lactose positive and gelatinase positive
What result will C. sporogenase give in a lactose gelatin medium?
Red liquid
Lactose negative
Gelatinase positive
What result will E.Coli give in a lactose gelatin medium?
yellow Gel
Lactose positive
Gelatinase negative
What is the principle behind the egg yok agar - Naglar reaction?
(4)
Used to detect lecithinase and lipase production and proteolytic activity of anaerobes -> recommended in the Naglar Test for the ID of C. perfringens
Egg yolk agar is used which acts as a supply of lecithin and free fats which allows detection of lecithinase and lipase production and proteolytic activity
Lecithin break down results in a white opaque zone of precipitation that spreads beyond the edge of the colony
Lipase activity forms an iridescent sheen on the surface of the agar
What is egg yolk agar?
(3)
A non-selective medium
Supplemented with a suspension of egg yolk and enriched with haemin and vitamin K
Haemin and vitamin K enhance the growth of anaerobic microorganisms
What is the Naglar test
A test used to differentiate C. perfringens from other perfringens -> detects the alpha toxin (lecithinase) produced by it
How does the Naglar test work
(2)
Works by neutralising lecithinase C produced through a specific antitoxin
It works by exploiting the lecithinase ability of the organism already seen on egg yolk agar -> antitoxin is added only to one half of the plate
What is a positive Naglar test for C. perfringens
Dissapearance of the cloudy lecithinase action
How do you carry out an Egg yolk -agar Naglar Test
(7)
Ask for Egg yolk agar, C. perfringens antitoxin and C. perfringens control
Split plate in two, label one half C. perfringens type A antitoxin
Swab half of plate with antitoxin A
Allow plate to dry
Inoculate egg yolk agar with a single streak across the plate starting from the side with no antitoxin
Do the same with + control C. perfringens
Incubate at 37 degrees for 48 hours
What is a positive lecithinase result
Appearance of opaque, diffuse zone that extends into the medium
What is a negative lecithinase result
Absence of a white, opaque zone
What is a positive lipase result
An irredescent sheen
What is a negative lipase result
Absence of irredescent sheen
What is a positive Naglar test result
Disappearance of opacity
What is a negative Naglar test result
No disappearance of opacity
