2nd test: Bacillus, Clostridium and GRAM staining Flashcards
GRAM staining
1st step
- Stain the smear with crystal violet for 3-5 minutes
GRAM staining
2nd step
- Pour off the stain, rinsing with tap water
GRAM staining
3rd step
- Stain the smear with Lugols iodine solution
for 1-1,5 minutes
GRAM staining
4th step
- Pour off the stain, rinsing with tap water
GRAM staining
5th step
- Differentiation: drop 5-6 drops of 96% ethanol on the smear, and let it flow down
GRAM staining
6th step
- Immediately rinse with tap water
GRAM staining
7th step
- Counterstaining with fuchsin or safranin
for 0,5-1 minute
GRAM staining
8th step
- Pour off the stain, rinsing with tap water
GRAM staining
9th step
- Drying with filter paper
GRAM staining
In which order do you apply which staining
1. crystal violet for 3-5 minutes
- Lugols iodine solution for 1-1,5 minutes
- drop 5-6 drops of 96% ethanol on the smear, and let it flow down
- Counterstaining with fuchsin or safranin
for 0,5-1 minute
Result of staining with GRAM STAINING of
GRAM + bacteria
Result: GR+ bacteria are blue,
Result of staining with GRAM STAINING of
GRAM - bacteria
GR- bacteria are red or pink,
Result of staining with GRAM STAINING of
Mycobacteria
mycobacteria are colorless,
Result of staining with GRAM STAINING of
Fungi
fungi are like GR+ bacteria.
What is Gram staining on histological slides called?
You can use Gram staining on histological slides as well, in this case the staining is called Brown-Brenn.
The background is yellowish brown, the color of the bacteria is like after a normal Gram staining.
GENUS: BACILLUS
Habitat
Diseased animals (VEGITATIVE)
Environment (SPORE)
GENUS: BACILLUS
MORPHOLOGY
4-5 micro.m ROD, CENTRAL SPORE
CAPSULE
NO FLAGELLA
GENUS: BACILLUS
RESISTANCE
Vegetative bacterium: Several days in carcass
(Skin: 2 weeks, Bonemarrow: 4 Weeks)
56 degrees in 15min
Spore: In soil can survive for more than 50 years
Boiling: 5-10min
Formalin (8-10%): 12-24 hours
GENUS BACILLUS: STAINING
GRAM-POSITIVE
TOLUIDINE-BLUE
(Metachromatic staining)
GENUS BACILLUS: CULTURE
Simple (Nutrient broth or agar, blood agar, air, 37 degrees)
R-S Colonies (depend on CO2)
Capsule: Poly-D-Glutamic acid (= INHIB PHAGOCYT.)
Virulence factor (= INHIB PHAGOCYT.)
Plasmid encoded
Spore: For spore formation
At least 15 degrees
Water
Oxygen
Cations are needed
= DO NOT OPEN CARCASS
37 degrees - finished during 16 hours
18 degrees it will start after 50 hours (2-3 days are needed)
GENUS BACILLUS: BIOCHEMISTRY/ANTIGENS
Active metabolism
Catalase +
Oxidase -
Ag: Capsule (poly-D-glutamic acid) - SAPHROPHYTES, Polysaccharide Hapten - Heat stabile
ASCOLI TEST, B.CEREUS - Cross reaction!
OEDEMA FACTOR
LETHAL FACTOR
PROTECTIVE ANTIGEN
ASCOLI THERMOPRECIPITATION TEST
- Detection of B.Anthracis heat stable cell wall Ag (Crossreacts with B.CERESUS)
GENUS BACILLUS: PATHOGENICITY
Mammals (mainly herbivorous)
GENUS BACILLUS: PATHOGENICITY
Cattle, Sheep
Fatal peracute or acute septicaemic anthrax.
GENUS BACILLUS: PATHOGENICITY
Pigs
Subacute ANTHRAX with oedematous swelling in the pharyngeal region, an intestinal form with higher mortality is less common
GENUS BACILLUS: PATHOGENICITY
Horses
Subacute ANTHRAX with localized oedema, septicemia with collic and enteritis sometimes occur
GENUS BACILLUS: PATHOGENICITY
Humans
Skin, Pulmonary and intestinal form of ANTHRAX
GENUS BACILLUS: PATHOGENICITY
CARNIVORES
Comparatively resistant
GENUS BACILLUS: PATHOGENICITY
BIRDS
Completely resistant
GENUS BACILLUS: PATHOGENICITY
Types of bacteria
-
B. Cereus
- Cattle:
Mastitis (Rare)
- Humans:
Food Poisoning (GI infections)
Eye infections
-
B. Licheniformis
- Cattle, Sheep
Sporadic Abortion
-
Paenibacillus larvae
- Honey bees
Amarican Foulbrood
GENUS BACILLUS: PATHOGENICITY
SAPHROPHYTE BACILLI
= Live on dead decaying matter
B. Subtilis
B. Megaterium
B. Cereus
B. Licheniformis: Bacitracin
B. Thuringiensis: Insect pathogen, pest control (moth)
Phaenibacillus (B.) Polymyxa: Polymyxins
Geobacillus (B.) Stearothermophhillus: Heat resistant test organism
GENUS BACILLUS: PATHOGENICITY
Most imp bacillus bacteria for vet med
- B. Anthracis
- B. Subtilis
- B. Megaterium
- B.Cereus
GENUS BACILLUS: PATHOGENICITY
Properties of B. ANTHRACIS
Flagella = NO
Capsule production in air = NO
Hemolysis = NO
Pathogenicity = YES
GENUS BACILLUS: PATHOGENICITY
Properties of B. SUBTILIS
Flagella = YES
Capsule production in air = YES
Hemolysis = NO
Pathogenicity = NO
GENUS BACILLUS: PATHOGENICITY
Properties of B. MEGATERIUM
Flagella = YES
Capsule production in air = YES
Hemolysis = NO
Pathogenicity = YES
GENUS BACILLUS: PATHOGENICITY
Properties of B. CEREUS
Flagella = YES
Capsule production in air = YES
Hemolysis = YES
Pathogenicity = YES
GENUS CLOSTRIDIUM - HABITAT
Soil
Mud
Water
Gut
GENUS CLOSTRIDIUM - MORPHOLOGY
7-10 micro.m ROD
Flagella (exc. C.perfr)
SPORE (Terminal, subterminal, central)
=Help identification
No capsule (exc. C, perfr)
GENUS CLOSTRIDIUM - RESISTANCE
Vegitative - (like GRAM +)
SPORE:
In DRY specimens: For years
- C. Botulinum : 3-4 hour boiling
- C. Perfr. spore: 5-10 min boiling, 8% formalin: 2 hours
GENUS CLOSTRIDIUM - STAINING
GRAM +
GENUS CLOSTRIDIUM - CULTURE
Anaerobic
Oxygen tolerance is different
(C. Tetani, C.Novyi B: Strickt)
(C. Histolyticum, C. Botulinum: Can tollerate O2)
Nutrient agar
Blood Agar
Optimal temp: 37 degrees
C. Perfringens: 40-45 degrees
C. Putrefaciens: 15-22 degrees
GENUS CLOSTRIDIUM - BIOCHEMISTRY/ ANTIGENS
CATALASE -
OXIDASE -
OF: FERMENTATIVE
Active metabolism, CARBOHYDRATES are fermented
(exc. C. Tetani, C. Histolyticum)
PROTEOLYTIC:
- C. tetani, C. Histolyticum, C. Botulinum
EXOTOXINS (toxin, toxoid or anatoxin, antitoxin)
Ag - Complex, close relationship
GENUS CLOSTRIDIUM - PATHOGENICITY
What are the different diseases caused by GENUS CLOSTRIDIUM
1. Gas gangrenic Diseases
- Malignant oedema (Cattle, Pig, Sheep)
- Black leg (Cattle, Sheep)
- Human gas gangrene
2. Enterotoxaemic Diseases
3. Intoxications
GENUS CLOSTRIDIUM - PATHOGENICITY
1. Gas Gangrenic Diseases
= Tissue death. Bacteria enter open wound: Cause gas to form beneath skin, leading to tissue death
Toxins also cause tissue death
MALIGNANT OEDEMA (Cattle, Pig, Sheep)
C. Septicum
C. Novyi
C. Haemolyticum
C. Histolyticum
C. Sordelli
Blackleg (Cattle, Sheep)
C. Chauvoei
Human Gas Gangrene
C. Perfringens A
C. Novyi A
GENUS CLOSTRIDIUM - PATHOGENICITY
- Enterotoxaemic Diseases
Necrotic ENTERITIS in CHICKEN
C.Perfringens A/C
LAMB DYSENTERY
C. Perfringens B
Necrotic ENTERITIS in PIGS, STRUCK of SHEEPS
C. Perfringens C
PULPY KIDNEY DISEASE of SHEEPS
C. Perfringens D
ULCERATIVE ENTERITIS of CHICKEN:
C. Colinum
GENUS CLOSTRIDIUM - PATHOGENICITY
- Intoxications
TETANUS = Rigid paralysis
C. Tetani
BOTULISM = FLACCID Paralysis
C. Botulinium
GENUS CLOSTRIDIUM - PATHOGENICITY
C. Septicum
Culture: Nutrient Agar
Toxins:
1- Alpha haemolytic= Necrotizing, Lethal
2- Beta Haemolytic = (DN-ase)
3- Gamma Haemolytic = (Hyaluronidase)
4- Sigma Haemolytic = (Haemolysin)
CATTLE, PIG, SHEEP = Malignant Oedema
SHEEP and CALVES= BRAXY (Abomastitis)
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
TOXIN TYPE A
- Necrotic Enteritis in Chickens
- Necrotizing enterocollitis in pigs
- Canine hemorrhagic enterocolitis
- Human gas gangrene, food infection
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
TOXIN TYPE B
- Lamb dysentery
- Haemorrhagic enteritis in calves and foals
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
TOXIN TYPE C
1. Necrotic Enteritis of Pigs
2. Struck in Sheeps
3. Necrotic enteritis in Chicken
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
TOXIN TYPE D
1. Pulpy kidney disease in sheep
2. Enterotoxaemia in calves, goats
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
TOXIN TYPE E
1. Haemorrhagic enteritis in calves
2. Enteritis in rabbits
GENUS CLOSTRIDIUM - PATHOGENICITY
All the bacterias
- C. Novyi
- C. Haemolyticum
- C. Histolyticum
- C. Chauvoei
- C. Perfringens
- C. Colinum
- C. Diffcile
- C. Tetani
- C. Botulinum
- C. Piliforme
- C. Spiroforme
GENUS CLOSTRIDIUM - PATHOGENICITY
C. NOVYI
- Toxins: Alpha, lipase, lecinase, DN-ase,
- CollagenaseTypes: A, B, C (Non-pathogenic, no toxins)
1. Gas gangrene:
Cl. Novyi A/B
2. Sheep, Cattle: Infectious necrotic hepatitis:
Cl. Novyi B
GENUS CLOSTRIDIUM - PATHOGENICITY
C. HAEMOLYTICUM
Cattle, Sheep
BACILLARY HAEMOGLOBINUIA
GENUS CLOSTRIDIUM - PATHOGENICITY
C. HISTOLYTICUM
Whide spread
Can tollerate O2
Toxins: Alpha, Collagenase, Protease, elastase, haemolysin
Cattle, Pig, Sheep: MALIGNANT OEDEMA
GENUS CLOSTRIDIUM - PATHOGENICITY
C. CHAUVOEI
Culture:
Blood agar, Strong Hemolysis, Only Guinea pigs are susceptible
Toxins:
As C. Septicum
Cattle, Sheep
Blackleg
Differentiation from C. Cepticum
Culture, IF, PCR, (Animal Trial, Toxin neutralization)
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PERFRINGENS
Optimal Temperature
40-45 degrees, No Flagella, have Capsule
Toxins:
Main: (alpha, beta, epsilon, iota - Activated by TRYPSIN)
Auxiliary: (Gamma, Delta, Theta, Kappa, Lambda, Mu) - Neutralization
GENUS CLOSTRIDIUM - PATHOGENICITY
C. COLINUM
Quail, Chicken, Turkey, Phesant:
ULCERATIVE ENTERITIS:
- Intestinal Ulceration,
- Hepatic Necrosis
GENUS CLOSTRIDIUM - PATHOGENICITY
C. DEFFICILE
Enterotoxin, Cytotoxin
Human
Pseudomembranaceus colitis
Foal
Hemorrhagic enterocolitis in newborn foals
Dog
Chronic Diarrhoea
GENUS CLOSTRIDIUM - PATHOGENICITY
C. TETANI
Strickt anaerobe
10 serotypes
Toxins:
Neurotoxins, rigid paralysis
Haemolysin
Fibrinolysin
Disease:
TETANUS
Horse, Man = Highly susceptible
Ruminant, Pig = Moderate susceptible
Carnivores = comparatively resistant
Birds = NOT SUSCEPTIBLE
GENUS CLOSTRIDIUM - PATHOGENICITY
C. BOTULINUM
Biochemically NOT UNIFORM
A-B-C-D-F-G Antigenically distinct toxin types
A, B, E, F
= Soil, activated by TRYPSIN - HUMAN DISEASE
C, D
= Soil, intestine - ANIMAL DISEASE
Toxin:
Heat stable (100 degrees - 20 min)
Inhibit the release of ACh in neuromuscular synapsis
1mg toxin = 30 000 000 mice LD50 (1200 tonnes mice)
GENUS CLOSTRIDIUM - PATHOGENICITY
C. PILIFORME
The systematic position is questionable
(obligate intracellular, Gram - variable, sporeforming filaments)
Culture
Mouse fibroblast, Liver
Embryonated EGG
Disease
TYZZER’s DISEASE
Foal
Under 6 weeks of age (dog, cat, rabbit)
SEVERE HEPATIC NECROSIS
GENUS CLOSTRIDIUM - PATHOGENICITY
C. SPIROFORME
Atypical COILED MORPHOLOGY
Rabbit: ENTERITIS
Pred factor:
Low fiber diet or AB Treatment
Iota-toxin production
Haemorrhagic Enteritis, Diarrhoea
FATAL WITHIN 48 HOURS
Epsilon- and iota toxins are activated by
trypsin!
Trypsin inactivates
Beta-toxin
Differentiation of the most important Bacillus sp.
FLAGELLA
NOT B. anthracis
Differentiation of the most important Bacillus sp.
CAPSULE (air)
ONLY
B. subtilis
B. megaterium
Differentiation of the most important Bacillus sp.
HEMOLYSIS
ONLY
B. cereus
Differentiation of the most important Bacillus sp.
PATHOGENICITY
ONLY
B.ANTHRACIS
B. cereus show strong β-haemolysis
True or false
True
What kind of clinical effect of tetanus
RIGID PARALYSIS
What kind of clinical effect in Botulism
FLACCID Paralysis