gram positive bacilli (rods) · gram stain of a smear from a culture of bacillus anthracis. most of...

Gram Positive Bacilli(Rods)

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AbD El-GAwAD M. HAsHEM

Classification of Gram Positive Bacilli

Non Spore Forming Bacilli– Pathogenic Member: E.g. C. Diptheriae– Non-Pathogenic Strains: E.g. diphtheroids

Non-Spore Forming Bacilli– Aerobic Spore Forming Bacilli

The Genus: Bacillus– Anaerobic Spore Forming Bacilli

The Genus: Clostridium

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Gram Positive Bacilli

I- Aerobic Non-Spore Forming Bacilli

Corynebacterium

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I- Basic Characters

Morphology– Shape: Pleomorphic gram positive Rods (generally

slender)– Non- spore forming, non capsulated.– Arrangement: arranged in angles thus show Chinese

letters apperance.– The Bacilli contain Polyphosphate (Voulten)

Granules usually at the PolesGram’s Stain: G+ve Bacilli (purple)Polychrome Methylene Blue: Reddish purple granules against lightly stained cytoplasm (Metachromatic Granules)

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Corynebacterium

Culture Characteristics– C.diptheriea is aerobe and facultative

anaerobe.– Blood Agar: See Image– Serum agar (Loeffelers serum): – Blood Tellurite agar: Potassium tellurite inhibit

the growth of most oral commensals has no effect on Corynibacterium. See images

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II- Microscopic examination

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Smear from a culture of a diphtheria bacillusThe club shape (thick at one end and tapering towards the other), The arrangement in so-called Chinese letter patterns,

Metachromatic granules of Corynebacterium

Gram stain ofCorynebacterium

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III- Culture characteristics

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Loffler’s seum mediumLoffler’s serum mediumNameHorse serum and nutrient broth 3:1 1% glucose

Constituents

EnrichedTypeGrowing and cultivation of C. diphtheriaUse

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Corynebacterium diphtheriae types on blood agar

Intermedius coloniesNon-haemolytic and are smaller than those of the other types.

Mitis colonies:Almost shiny smooth surface. Colonies surrounded by a narrow zone of haemolysis.

Gravis colonies:Large With dull matt surface faint radial Striations at the Periphery. Some gravis strains are haemolytic,

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C. diphtheriae On McLeod’s tellurite medium

Intermedius coloniesMuch smaller than those of gravis or mitis. Typical colonies, with their dark centres and lighter margins, have been likened to frog’s eggs.

Mitis colonies:Have a black raised centre which slopes gently down to a pale rim. This is the ‘coolie-hat’ colony which is common among mitis strains.

Gravis colonies:flattened cones with metallic grey centres, Radially striated margins. ‘daisy head’ colonies on McLeod’s medium.

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IV-Serological Reactions

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Elek’s Test For Toxigenicity Of Diphtheria Bacilli

It is an in vitro method for identifying a toxigenic strain of diphtheria bacillus. (Elek’s medium, 2 days at370C, then 2 days at 40C; transmitted light, xi.)Suspected strain is streaked across the plateFilter paper strip saturated with antitoxin is placed at right angle. Toxigenic strains develope lines of precipitate in the zone of optimal antigen-antibody proportion.

In Elek’s medium, which favours the Production of diphtheria toxin.

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Elek’s TestElek’s testName20% horse serum + agarConstituentsToxin from M.O. meets antitoxin (antibody) from filter paper forming precipitation lines

Principle

Differentiation between toxigenic & non-toxigenic strains of C. diphtheria

Use

Gel precipitation reactionType of reaction

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II- Aerobic Spore Forming Bacilli

Bacillus anthracis

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Gram stain of a smear from a culture ofBacillus anthracis

Most of the cells are arranged in chains, although there are a few singles and pairs. The bacilli are large and gram-positive.

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Bacillus anthracis in a blood smear

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Spores of Bacillus anthracis

This smear is from an older culture , The spores are stained green and the vegetative cells red. Some of the spores are still within the cells, but others are free. Note that the cell bodies are not distended by the spores

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Biochemical Reactions

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Hydrolysis of gelatin (Gelatin liquefication)

After incubation these tubes were stood in crushed ice for one hour, and then tilted to find which had gelled. From left to right:Bacillus anthracis, negative reaction for hydrolysis of gelatin Bacillus cereus, positive reaction.

Gelatin dissolves in warm water and gels when cool. If it is hydrolysed, it does not gel, but remains liquid when cooled.

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GelatinaseNameNutrient broth + gelatin (12-15%)Constituents

Gelatin is liquified by M.O. producing gelatinasePrincipleTest for gelatinase producing M.O. e.g. Bacillus, Pseudomonas or Staphylococcus

Use

Hydrolysis of gelatin (Gelatin liquefication)

Positive

Negative

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Hydrolysis of starch (Amylase test)

1. Inoculate a plate of nutrient agar containing 1% starch by streaking with the tested culture.2. Incubate at 37°C for 48 h. Add iodine solution. The appearance of a colourless zone around the streaking indicates starch hydrolysis.

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Hydrolysis of starch (Amylase test)AmylaseNameNutrient agar + 1% soluble starchConstituents

Starch is hydrolysed by amylase enzyme.After addition of iodin a blue color develops except around growth

Principle

Test for amylase producing M.O. e.g. Bacillus, Pseudomonas or Staphylococcus

Use

Positive Negative

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III- Anaerobic Spore Forming Bacilli

Clostridium

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Classification of Genus ClostridiaPathogenic Closridia can be classified into

Clostridia of wound infection– Clostridium causing gas Gangrene

Saccharolytic–C.perfringens, C.novyi, C.septicum

Proteolytic–C.histoliticum, C.sporogees, C.perfringens

– Clostridium TetaniEnteropathogenic clostridia.– C. Botulinum

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I- Basic CharactersMorphology

Most of genus Clostridia show the following:– Shape: gram positive Rods– spore forming, Motile.– Arrangement: Single or in pairs

C. perfringens:– Some cells appears gram – ve especially in old cultures– In sugar media rods are shorter. In Protien rich media cells may become

filamentous.– Non motile and Spores formed in small no.

C. tetani:– Charctarized with spherical terminal spore giving the typical drum stick

apperance.

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II- Microscopic examination

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Gram stain Of Cl. Perfringensfrom gas gangrene

Note the necrotic background, without intact inflammatory cells or muscle cells, and relatively large “box car”-shaped gram-positive rods of C. perfringens

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Stained smears of Clostridium tetani

Negatively stained (Nigrosin): The terminal spores when mature are spherical and of much greater diameter than the vegetative cells. The ‘drum-stick’ morphology is characteristic of this species.

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Differential staining by fluorescent antibodies

Fluorescent antibody methods allow rapid identification of bacteria and other micro-organisms in smears made either directly from infected tissues or from cultures.A smear containing a mixture of two clostridum strains was treated with an two different antibody labeled with fluorescent dyes.Then examined by fluorescence microscopy.

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III- Culture characteristics.

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Anaerobic cultivation

Anaerobic jar for cultivation of anaerobic microorganisms.

Gas bags are used to remove oxygen for the jar

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C. perfringens on blood agar

Note the double zone of hemolysis. The inner zone of complete hemolysis is due to theta-toxinouter zone of incomplete hemolysis to alpha-toxin (lecithinase activity).

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Cultures of Clostridium on cooked meatCultures of, left to r ight, Clostr idium welchii, C. sporo genes and C. tetaniinThe proteolytic C. sporogenes has turned the meat black, and has digested the sharp edges off the meat granules. (In the laboratory the most obvious feature of this species is its putrid odour.) All three species have produced much gas; in two this has blown the plug of meat particles up the tube.

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Biochemical reactions

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Nagler’s reactionLecithinase testName

Serum or egg yolk medium (contain lecithin, Ca and Mg),Constituents

Colonies are surrounded by an opalescent zone of precipitated lipid released from lecithin in the egg yolk by lecithinase activity.

Principle

identification of C. perfringenes (welchiUse

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Action of clostridia on litmus milk

1. Screw capped tube of Litmus Milk is inoculated with the tested culture.

2. Incubate anaerobically at 37°C for 48h.3. After incubation, the tube is examined for acid, gas and

stormy clot production.C. tetani, produced acid C. welchii, produced a clot which has been disrupted by gas the typical ‘stormy clot’.

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Litmus milk

Litmus milk1) neutral 2) acid 3) alkaline

1) neutral

2) acid3) alkaline

Litmus milkName

Skimmed milkConstituents

LitmusIndicator

Some M.O. have proteolytic activity Casein gives alkaline end products

Principle

(differential medium)Type

Differentiation bet. Cl. Perfringenes (saccharolytic) andCl. Tetani (proteolytic)

Use

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gram positive bacilli (rods) · gram stain of a smear from a culture of bacillus anthracis. most of...

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