Practical G- rods

Lab : 9 Gram (–)ve rods 1- Enteric rods Facultative an-aerobic (Enterobacteriacea) 2- non - Enteric rods Obligatory aerobic Facultative an-aerobic a. Facultative an-aerobic(Enterobacteriacea) * Microscopic examinationShort G - ve rods , motile (except klebsiella and shigella), non –capsulated (except klebsiella) Cultural appearance *the colonies of enterobacteriacea in general (circular ,convex ,smooth colonies ).On MacConkey : Selective media for enterobacteriacea . Differential media *Lactose fermenter (pink colonies) such as E.coli and Klebsiella. *Non- lactose fermenter (colorless colonies) such as Proteus , Salmonella and Shigella.

Escherichia coli

It is found as normal flora in large intestine of most humans , though usually benign , some strains cause traveler's diarrhea , even benign strains can cause urinary tract infectionsMicroscopic examination Gram ( - ) ve rods ,straight with rounded ends ,mostly are motile . Cultural appearance *Nutrient agar : Opaque , smooth,convex,with entire edges .Blood agar : some produce β –hemolysis . Eosin methylene blue ( EMB ) : metalic green sheen .Mac Conky agar : pink (lactose Fe.) , non viscous colonies .Biochemical testٍ (TSI) test : Acid slant (+)ve* Acid butt (+)ve With gas production , but no H2S produced .IMVC : + + - -Urease : ( - ) veNitrate reduction : (+)ve

metalic green on ( EMB )

left: no lactose fermentation right: lactose fermentation
Differential Media:
MacConkey Agar

Klebsiella

It is found as normal flora (large intestine , urethra) , human feces, soil, water, grain, fruits, vegetables , may cause pediatric septicemia , pneumonia , urinary tract infections Microscopic examination Gram ( - ) ve rods , capsulated , non motile . Cultural appearance * Blood agar : gray, large colonies , circular , entire, high convex, often mucoid on primary isolation.. Mac Conky agar : usually pink in center with clear edge (lactose Fer.) Biochemical test (TSI) test : Acid slant (+)ve* Acid butt (+)ve With gas production no H2S produced IMVC : - - + + Urease :( + ) ve Nitrate reduction : (+) ve

Klebsiell

Proteus



Biochemical test (TSI) test : alk slant (-) or Acid slant (+)ve for P vulgaris * Acid butt (+)ve With gas production , H2S produced . IMVC : v + - v Urease : (+) ve Nitrate reduction : (+) ve Phenylalanine deaminase test : (+) ve

Proteus vulgaris

Salmonella

Transmitted from animal to human by the oral rout ( milk ) ,it result in enteritis systemic infection ,food poisoning ( Salmonella typhimurium) , typhoid fever (Salmonella typhi ) , or para typhoid fever (Sal. paratyphi A , Sal. paratyphi B) .* Microscopic examinationGram ( - ) ve rods Cultural appearance*circular ,smooth , entire edge ,.Mac Conky agar : pale colonies (non lactose fer.).●Bismuth Sulfite agar ( BSA ) : black to brown colonies with metallic sheen appearance , because of H2S production .Salmonella Shigella agar (SSA) : colorless colonies with black centers .

Bismuth sulfate agar is selective for Salmonella spp.

Widal test(indirect latex agglutination test)
Is used to detect antibodies against H and O antigens of the Salmonella typhi and paratyphi. The significant Abs titer for both anti-O and anti-H is 1/160 or more.

Shigella

Biochemical test (TSI) test : Alk slant (-) ve * Acid butt (+) ve No gas production , no H2S produced . IMVC : v + - - Urease : (-) ve Nitrate reduction : (+) ve

G G+ve rods

Tetanus e.g. Cl. tetani
Non spore forming
Spore forming o2 co2
corynebacterium Diphtheria
Bacillus anthracis
Clostridium perfringens
Bacillus subtilis
Cl. botulinum

Clostridium tetani
Cl. difficille
Bacillus cereus


Corynebacterium diphtheriae
Microscopic examination * Gram +ve rods * pleomorphic (Chinese letter,V, palisades arrangment) * Non spore forming * non motile * non capsulated * Metachromatic granules (volutin granules) * Blue biplar with albert stain

Macroscopic appearance

Cultural appearance Black colony on tellurite blood agar (Tinsdales agar) due to the production of H2S from cystine biochemical test Gelatin -ve Oxidase +ve Starch +ve Glucose +ve Maltose +ve

Bacillus anthracis

Bacillus anthracis is a pathogenic bacterium that causes anthrax disease Microscopic examination* Gram +ve rodsArranged in long chains with squre endsaerobicOval central spore , not bulgingCapsulatedNon motile Macroscopic appearanceCultural appearanceϒ-hemolysis on blood agarMedusa head on nutreint agar

biochemical test Gelatin +ve Catalase +ve Medusa head of bacillus anthracis

Bacillus subtilis
Microscopic examination Gram +ve rods blunt ends Oval central or subterminal spore,bulging Motile Non Capsulated

Macroscopic appearanceCultural appearanceβ- hemolysis on blood agarMedusa head on nutreint agar biochemical testGelatin +veCatalase +ve

Clostridium

Cl. tetani Tetanus
Cl. Perfringens Gas gangrene
Cl. botulinum Botulism
Cl. difficille Antibiotic associated diarrhea

Cl. Perfringens

Cl. Perfringens( C. welchii ) is a pathogenic bacterium that causes Gas gangrene and Food poisoning (Enterotoxin) Microscopic appearance Large Gram-positive bacilli with blunt ends The spore is oval, sub-terminal & non bulging Spores are rarely observed Anaerobic Capsulated Non motile

Macroscopic appearanceCultural appearanceOn blood agar → • double zone of hemolysis on blood agar in cooked meat medium (prepared from heart muscles) which contain hematin & glutathione that act as reducing agent → pink due to saccharolytic prcess

biochemical test Nagler reaction is +ve The Nagler test is a biochemical test that is used to identify organisms which liberate phospholipases (lecithinases) e.g. Clostridium perfringens. The alpha toxin of C. perfringens has phospholipase activity and hence, when grown on a medium containing egg yolk phospholipid, the organism can break down this insoluble triglyceride. Phosphoryl choline release is seen as an area of opacity around the bacterial colonies . Antitoxin can inhibit a positive response

Nagler Reaction

Positive Nagler Reaction
Procedure of Nagler Reaction

Clostridium tetani

Microscopic appearance Gram positive, slender rod with rounded ends spherical terminal spore as drumstick Obligate anaerobe Motile Non capsulated Iodine (1%) in water is able to kill the spores within a few hours

Macroscopic appearanceCultural appearance* On blood agar : α- hemolysis → later β-hemolysis * in cooked meat medium(prepared from heart muscles) which contain hematin & glutathione that act as reducing agent → blackening of meat will observed with the production of H2S and NH3 (proteolytic ) biochemical testGelatin +ve

Cl. botulinumMicroscopic appearance

Gram positive with rounded ends form oval central or subtermminal endospore,bulginganaerobic MotileNon capsulatedMacroscopic appearanceCultural appearancein cooked meat medium → blackening of meat will observed with the production of H2S and NH3 (proteolytic )

Clostridia

Large Gram positive Straight or slightly curved rods with slightly rounded ends Anaerobic bacilli Spore bearing Spore do not germinate and growth does not normally proceed unless a suitably low redox potential Eh exists Saprophytes Some are commensals of the animal & human gut which invade the blood and tissue when host die and initiate the decomposition of the corpse (dead body) Causes diseases such as gas gangrene, tetanus, botulism & pseudo-membranous colitis by producing toxins which attack the neurons pathways

Clostridia of medical importance

Clostridium Causing
Tetanus e.g. Cl. tetani
Gas gangrene
Botulism e.g. Cl. botulinum
Saccharolytic e.g. Cl. perfringens &Cl. septicum
Proteolytic e.g. Cl. sporogenes
ِAntibiotic associated diarrheae.g. Cl. difficille Mixed: Cl. histolyticum

Clostridium Causing TetanusCl. tetani

Micrscopic apperance Gram positive, slender rod with rounded ends All species form endospore (drumstick with a large round end) Obligate anaerobe Motile Grows well in cooked meat broth and produces a thin spreading film when grown on enriched blood agar Iodine (1%) in water is able to kill the spores within a few hours

Toxins

Cl. tetani produces two types of toxins: Tetanolysin, which causes lysis of RBCs Tetanospasmin is neurotoxin and essential pathogenic product Tetanospasmin is toxic to humans and various animals when injected parenterally, but it is not toxic by the oral route Tetanospasmin which causes increasing excitability of spinal cord neurons and muscle spasm

Laboratory Diagnosis of Tetanus

The diagnosis of tetanus depends primarily upon the clinical manifestation of tetanus including muscle spasm and rigidity. Specimen: Wound exudates using capillary tube Culture: On blood agar and incubated anaerobically Growth appears as a fine spreading film. Gram stain is a good method for identifying Clostridium Cl. tetani is Gram positive rod motile with a round terminal spore giving a drumstick appearance


Clostridium Causing Gas Gangrene
Clostridia causing gas gangrene
Saccharolytic organisms Cl. perfringens, Cl. septicum Ferment carbohydrates Acid and gas are produced
Proteolytic organisms Cl. sporogenes Digest proteins with blackening bad smell production
Mixed saccharolytic & proteolytic Cl. histolyticum

Saccharolytic Microorganisms

Clostridium perfringens
Large Gram-positive bacilli with stubby ends Capsulated Non motile (Cl. tetani is motile) Anaerobic Grown quickly on selective media Can be identified by Nagler reaction

Toxins

The toxins of Cl. perfringens  toxin (phospholipase C, lecithinase) is the most important toxinLyses of RBCs, platelets, leucocytes and endothelial cellsIncreased vascular permeability with massive hemolysis and bleeding tissue destructionHepatic toxicity and myocardial dysfunction-toxin is responsible for necrotic lesions in necrotizing enterocolitisEnterotoxin is heat labile toxin produced in colon → food poisoning

Laboratory Diagnosis

Specimen: Histological specimen or wound exudatesHistological specimen transferred aseptically into a sterile screw-capped bottle & used immediately for microscopical examination & culture Specimens of exudates should be taken from the deeper areas of the wound where the infection seems to be most pronounced Microscopical examination (Gram, Spore stain etc)Gram-positive bacilli, non motile, capsulated & sporulated The spore is oval, sub-terminal & non bulgingSpores are rarely observedCulture: Anaerobically at 37COn Robertson's cooked meat medium → blackening of meat will observed with the production of H2S and NH3On blood agar → β-hemolytic colonies

Biochemical Tests

Cl. perfringnes characterized by: It ferments many carbohydrates with acid & gas It acidified litmus milk with stormy clot production Nagler reaction is positive

Reaction on Litmus Milk

Litmus Milk
Skimmed Milk (Without Fat)
Litmus indicator
Acid Base and Redox indicator
Lactose Sugar
Casein Protein
Contains

Reaction on Litmus Milk

Lactose Acid Pink Color (Milk Sugar)
Fermentation
Litmus Indicator
1- Acidic Reaction
2- Basic Reaction
Casein Alkaline amines Blue Color (Milk Protein)
Digestion
Litmus Indicator

Reaction on Litmus Milk

Stormy Clot Formation
Fermentation
Casein Milk Protein
Coagulation
Gas
Clot
Stormy Clot
Milk Sugar
Lactose
Acid
+

Reaction on Litmus Milk

Nagler’s Reaction This test is done to detect the lecithinase activityThe M.O is inoculated on the medium containing human serum or egg yolk (contains lecithin)The plate is incubated anaerobically at 37 C for 24 hColonies of Cl. perfringens are surrounded by zones of turbidity due to lecithinase activity and the effect is specifically inhibited if Cl. perfringens antiserum containing  antitoxin is present on the medium

Anaerobic Cultivation

Removal of oxygen & replacing it with inert gas Anaerobic Jar It is especially plastic jar with a tightly fitted lid Hydrogen is introduced from commercially available hydrogen generators envelop 10 ml of water is added to envelop immediately before placing it in the jar Hydrogen and carbon dioxide will release and react with oxygen in the presence of catalyst to form water droplet Anaerobic indicator (Methylene blue) is placed in the jar Methylene blue is blue in oxidized state (Aerobic condition) while turns colorless in reduced state (Anaerobic condition)


Anaerobic Cultivation
Culture Media containing reducing agent Thioglycollate broth It contains Sodium thioglycollate (Reducing agent) Rezazurin (redox indicator) Low percentage of Agar-Agar to increase viscosity of medium Cooked Meat Medium It contains Meat particles (prepared from heart muscles) which contain hematin & glutathione that act as reducing agent

Growth on Fluid Thioglycolate

Clostridium sporogenes Growing in Thioglycolate Medium
Reducing agents in the medium absorb oxygen and allow obligate anaerobes to grow

Reaction on Cooked Meat Medium

Saccharolytic reaction It causes fermentation of glycogen of muscles Production of acid and gas Meat particles remain intact e.g Cl. perfergines Proteolytic Reaction It causes digestion of meat particles Formation of black, foul smelling due to sulfur compounds

Anaerobic Jar

Candle Jar