Facultative an-aerobic a. Facultative an-aerobic(Enterobacteriacea) 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 examination Short 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 infections Microscopic 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 test ٍ (TSI) test : Acid slant (+)ve* Acid butt (+)ve With gas production , but no H2S produced . IMVC : + + - - Urease : ( - ) ve Nitrate reduction : (+)ve

MacConkey Agar Differential Media: metalic green on ( EMB ) left: no lactose fermentation right: lactose fermentation Differential Media: MacConkey Agar metalic green on ( EMB )

Klebsiella Microscopic examination Acid butt (+)ve With gas production 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 * Microscopic examination Cultural appearance It is found as normal flora (large intestine , urethra), manure; soil; polluted waters, Only cause infection when leave intestinal tract (Opportunist) may cause urinary tract and wound Infection (especially burns) ; infantile diarrhea   * Microscopic examination Gram ( - ) ve rods , it is highly motile . Cultural appearance Fishy smell and swarming appearance. Blood agar : an irregular flat colony that can rapidly spread across the surface of a fresh plate . Mac Conky agar : flat irregular colorless colonies, may spread on fresh medium .

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 * Microscopic examination 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 examination Gram ( - ) 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 .

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

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 It is normal flora (large intestine),may cause bacillary dysentery (shigellosis ); traveler's diarrhea. S. dysenteriae is the agent of classic bacterial dysentery   * Microscopic examination Gram ( - ) ve rods ,non motile Cultural appearance * smooth ,convex , entire edge , transparent . Mac Conky agar : pale colonies (non lactose fer.). Salmonella Shigella agar (SSA) : colorless colonies without black centers .

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

Clostridium perfringens G G+ve rods Tetanus e.g. Cl. tetani Non spore forming Spore forming o2 co2 Bacillus anthracis corynebacterium Diphtheria Bacillus subtilis Bacillus cereus Clostridium perfringens Clostridium tetani Cl. botulinum Cl. difficille

Corynebacterium diphtheriae Corynebacterium diphtheriae is a pathogenic bacterium that causes diphtheria. It is also known as the Klebs-Löffler bacillus 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 Microscopic examination * Gram +ve rods Bacillus anthracis is a pathogenic bacterium that causes anthrax disease Microscopic examination * Gram +ve rods Arranged in long chains with squre ends aerobic Oval central spore , not bulging Capsulated Non motile Macroscopic appearance Cultural appearance ϒ-hemolysis on blood agar Medusa 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 appearance Cultural appearance β- hemolysis on blood agar Medusa head on nutreint agar biochemical test Gelatin +ve Catalase +ve

Clostridium Cl. tetani Tetanus Cl. Perfringens Cl. botulinum Botulism 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 appearance Cultural appearance On 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 appearance Cultural 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 test Gelatin +ve

Cl. botulinum Microscopic appearance Gram positive with rounded ends form oval central or subtermminal endospore,bulging anaerobic Motile Non capsulated Macroscopic appearance Cultural appearance in 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 Cl. 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

Saccharolytic Microorganisms Cl. perfringens Causing Gas gangrene (Enterotoxin) Food poisoning

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 toxin Lyses of RBCs, platelets, leucocytes and endothelial cells Increased vascular permeability with massive hemolysis and bleeding tissue destruction Hepatic toxicity and myocardial dysfunction -toxin is responsible for necrotic lesions in necrotizing enterocolitis Enterotoxin is heat labile toxin produced in colon → food poisoning

Laboratory Diagnosis Specimen: Histological specimen or wound exudates Histological 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 bulging Spores are rarely observed Culture: Anaerobically at 37C On Robertson's cooked meat medium → blackening of meat will observed with the production of H2S and NH3 On 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 Contains Skimmed Milk (Without Fat) Litmus indicator Lactose Sugar Casein Protein Acid Base and Redox indicator

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

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

Reaction on Litmus Milk

Nagler’s Reaction This test is done to detect the lecithinase activity The 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 h Colonies 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 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

Candle Jar Anaerobic Jar