1. Facultative an-aerobic a. Facultative an-aerobic(Enterobacteriacea)
Lab : 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.

2. 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

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

5. 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

6. Klebsiell

7. 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 .

8. 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

9. 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 .

10. 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

11. Bismuth sulfate agar is selective for Salmonella spp.

12. 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.

13. 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 .

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

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

22. 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

23. 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

24. 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

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

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

27. Macroscopic appearance Cultural appearance β- hemolysis on blood agar
Medusa head on nutreint agar
biochemical test
Gelatin +ve
Catalase +ve

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

29. 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

30. 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

31. 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

32. Nagler Reaction
Positive Nagler Reaction
Procedure of Nagler Reaction

33. 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

34. 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

35. 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 )

38. 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

42. Clostridia of medical importance

43. 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

44. 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

45. 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

46. Clostridium Causing Gas Gangrene

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

48. 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

49. 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

50. 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

51. 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

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

53. 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)

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

55. Reaction on Litmus Milk

56. 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

57. 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)

58. 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

59. Growth on Fluid Thioglycolate
Clostridium sporogenes Growing in Thioglycolate Medium
Reducing agents in the medium absorb oxygen and allow obligate anaerobes to grow

60. 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

61. Candle Jar
Anaerobic Jar