Clostridium species on blood agar as shown above picture. The name Clostridium is derived from the word ‘Kloster’ meaning a spindle. Most species of this genus, Clostridium are saprophytes found in soil, water, and decomposing plant and animal matter whereas Cl. tetani and Cl. perfringens are commensals of the intestinal tract of humans and animals.#
Clostridia are Gram-positive rods, motile ( except Cl. perfringens and Cl. tetani type VI), non-capsulated ( except Cl. perfringens and Cl. butyricum), obligate anaerobes capable of producing spores that protect them in a harmful environment. Individual cells are rod shaped. The spores are usually wider than the rods and are located terminally or sub terminally. Most clostridia are motile by peritrichous flagella while others have capsule-like Clostridium perfringens characteristics. Clostridium consists of around 100 species that include common free-living bacteria as well as important pathogens There are five main species responsible for disease in humans. Cl. perfringens: gas gangrene; food poisoning C. tetani: tetanus, C. botulinum: botulism C. difficile: pseudomembranous colitis, and Cl. sordellii : can cause a fatal infection in exceptionally rare cases after medical abortions.
Clostridia are Gram-positive, spore-forming rods, usually having sizes of 3-8 X 0.4-1.2 µm. They are highly pleomorphic. The shape and position of spores vary in different species and are useful for the identification of Clostridia. The spores may be at central in Cl. bifermentans , sub-terminal in Cl. perfringens , Oval and terminal in Cl. tertium, and spherical and terminal giving drum stick appearance in Cl. tetani.
Cl. perfringens is a relatively large Gram-positive short fat bacilli with blunt ends. It is encapsulated and non-motile. Anaerobic. It grows quickly on laboratory media on blood agar ( beta-hemolytic ) and has the following virulence factors – toxins, alpha-toxin – causes RBC rupture, edema, and tissue destruction, enterotoxin, collagenase, and hyaluronidase.
The anaerobic bacterium of the genus species Clostridium and is gram positive, slender bacillus and it has spherical terminal spores giving a drum stick appearance It is non encapsulated and motile with peritrichous flagella. It produces a potent biological toxin, tetanospasmin, and is the causative agent of tetanus a disease characterized by painful muscle spasms that can lead to respiratory failure and, in up to 40% of cases, death.
The worm botulism means sausage as the poorly cooked sausage was formerly associated with this type of food poisoning. This organism causes a severe form of food poisoning. It is a gram-positive rod, non-encapsulated, motile with peritrichate flagella, and produces sub-terminal, oval, bulging spores.
It was first isolated from the feces of newborn infants and so named due to unusual difficulties involved in the isolation of this pathogen. It is a long, slender, Gram-positive rod containing oval and terminal spores.
It depends on the involvement of species of the genus, Clostridium and they are as follow-
The diagnosis should always be made clinically and laboratory tests are done to confirm it. Laboratory diagnosis may be made by demonstrating bacilli by microscopy, culture, or by animal inoculation. Specimens generally collected are wound swabs, exudate, or tissue from the wound.
1. Microscopy
Gram stain is a very useful tool for screening the genus and helpful for presumptive identifications of Clostridium species.
Requirements for Gram stain
a) Compound light microscope
b) Reagents and glasswares
c) Quality control strains
Positive Control (PC) : Staphylococcus aureus (ATCC 25923)
Negative Control (NC): Escherichia coli (ATCC 25922)
d) Specimen ( Note:-5% sheep blood agar having growth of Clostridium species used and growth of this organism in thioglycollate broth or Robertson’s cooked meat medium can also be used.)
Preparation of bacterial smear: from liquid culture
Preparation of bacterial smear: from the solid medium
Procedure
Observation
Positive Control: violet color, round in shape in single, pairs and cluster
Test: red color and rod in shape
Negative Control: red in color and rod in shape
Result and Interpretation of Gram Stain
Gram-positive: purple or violet color
Gram-negative: Pink or red in color
Cocci: round in shape
Bacilli: rod in shape
Positive Control(PC): Gram-positive cocci in single, pairs and cluster
Test: Gram-positive bacilli or rods and also with evidence of spores as shown above picture
Negative Control(NC): Gram-negative bacilli
Liquid media for cultivation of anaerobes are –
Solid medium for cultivation of anaerobes are-
Principle
Mechanism of anaerobiosis achieves by following methods-
Displacement of oxygen
Absorption of oxygen
Combustion of oxygen or their combinations
Deep nutrient agar tube is the simplest method. Anaerobes grow in the depth of the medium, and the number of colonies becomes fewer towards the surface. Strict anaerobes will not grow within a centimeter of the surface.
Using reducing agents like glucose, ascorbic acid, cysteine, thioglycollate in the medium. Cooked meat particle also acts as s good reducing agent due to having glutathione e.g. Robertson’s cooked meat medium.
Another simple method of anaerobiosis in the application of a candle jar. A burning candle can not continue due to the exhaustion of oxygen inside the jar.
In laboratories, combustion involves the combining of oxygen with hydrogen to form water in the presence of a catalyst like palladium or palladinized asbestos. Anaerobic jars are a constant feature of anaerobic culture. They include the McIntosh and Fildes anaerobic jar, which has ilets to admit hydrogen and carbon dioxide, a vacuum pump for evacuating oxygen, and a catalyst fitted into the lid.
Similarly, a simpler but more expensive technique is the Gaspak system. This utilizes a transparent polycarbonate jar with a lid bearing a screened catalyst chamber. The catalyst, consisting of pellets of sodium borohydride, cobalt chloride, citric acid, and sodium bicarbonate is contained in the sachet. Water is added to the sachet and it is immediately placed in the jar, which is then sealed tightly. The resulting reaction liberates hydrogen and carbon dioxide. An indicator also adds to demonstrate anaerobiosis.
Requirements
Procedure
Quality control
Pseudomonas aeruginosa does not grow anaerobically.
Clostridium sporogenes do not grow aerobically.
Observations
When anaerobiosis is complete, obligate anaerobes like Clostridium sporogenes will grow, while obligate aerobes like Pseudomonas aeruginosa will not grow.
Result and Interpretation
Test for Cl. perfringens
The commonly available methods are-
It also depends on the involvement of the organism and the type of infection. Antibiotics useful for the treatment of gas gangrene are metronidazole, penicillin, sulphonamides, tetracycline, and amoxicillin. Tetanus treatment consists of controlling spasms, maintaining the airway by tracheotomy, and attention to feeding. The antitoxin may be used to neutralize the unbound toxins. Antimicrobial therapy with penicillin or metronidazole should be started and continued for a week or more. Similarly, for the treatment of Cl. difficle, metronidazole is the choice of drug.