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Bacto practical > 2nd test: Bacillus, Clostridium and GRAM staining > Flashcards

2nd test: Bacillus, Clostridium and GRAM staining Flashcards

1
Q

GRAM staining
1st step

A
  1. Stain the smear with crystal violet for 3-5 minutes
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2
Q

GRAM staining
2nd step

A
  1. Pour off the stain, rinsing with tap water
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3
Q

GRAM staining
3rd step

A
  1. Stain the smear with Lugols iodine solution

for 1-1,5 minutes

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4
Q

GRAM staining
4th step

A
  1. Pour off the stain, rinsing with tap water
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5
Q

GRAM staining
5th step

A
  1. Differentiation: drop 5-6 drops of 96% ethanol on the smear, and let it flow down
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6
Q

GRAM staining
6th step

A
  1. Immediately rinse with tap water
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7
Q

GRAM staining
7th step

A
  1. Counterstaining with fuchsin or safranin

for 0,5-1 minute

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8
Q

GRAM staining
8th step

A
  1. Pour off the stain, rinsing with tap water
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9
Q

GRAM staining
9th step

A
  1. Drying with filter paper
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10
Q

GRAM staining
In which order do you apply which staining

A

1. crystal violet for 3-5 minutes

  1. Lugols iodine solution for 1-1,5 minutes
  2. drop 5-6 drops of 96% ethanol on the smear, and let it flow down
  3. Counterstaining with fuchsin or safranin

for 0,5-1 minute

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11
Q

Result of staining with GRAM STAINING of

GRAM + bacteria

A

Result: GR+ bacteria are blue,

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12
Q

Result of staining with GRAM STAINING of

GRAM - bacteria

A

GR- bacteria are red or pink,

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13
Q

Result of staining with GRAM STAINING of

Mycobacteria

A

mycobacteria are colorless,

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14
Q

Result of staining with GRAM STAINING of

Fungi

A

fungi are like GR+ bacteria.

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15
Q

What is Gram staining on histological slides called?

A

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.​

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16
Q

GENUS: BACILLUS

Habitat

A

Diseased animals (VEGITATIVE)

Environment (SPORE)

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17
Q

GENUS: BACILLUS

MORPHOLOGY

A

4-5 micro.m ROD, CENTRAL SPORE

CAPSULE

NO FLAGELLA

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18
Q

GENUS: BACILLUS

RESISTANCE

A

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

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19
Q

GENUS BACILLUS: STAINING

A

GRAM-POSITIVE

TOLUIDINE-BLUE

(Metachromatic staining)

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20
Q

GENUS BACILLUS: CULTURE

A

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)

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21
Q

GENUS BACILLUS: BIOCHEMISTRY/ANTIGENS

A

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)
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22
Q

GENUS BACILLUS: PATHOGENICITY

A

Mammals (mainly herbivorous)

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23
Q

GENUS BACILLUS: PATHOGENICITY

Cattle, Sheep

A

Fatal peracute or acute septicaemic anthrax.

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24
Q

GENUS BACILLUS: PATHOGENICITY

Pigs

A

Subacute ANTHRAX with oedematous swelling in the pharyngeal region, an intestinal form with higher mortality is less common

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25
Q

GENUS BACILLUS: PATHOGENICITY

Horses

A

Subacute ANTHRAX with localized oedema, septicemia with collic and enteritis sometimes occur

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26
Q

GENUS BACILLUS: PATHOGENICITY

Humans

A

Skin, Pulmonary and intestinal form of ANTHRAX

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27
Q

GENUS BACILLUS: PATHOGENICITY

CARNIVORES

A

Comparatively resistant

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28
Q

GENUS BACILLUS: PATHOGENICITY

BIRDS

A

Completely resistant

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29
Q

GENUS BACILLUS: PATHOGENICITY

Types of bacteria

A
  1. B. Cereus
    - Cattle:

Mastitis (Rare)

  • Humans:

Food Poisoning (GI infections)

Eye infections

  1. B. Licheniformis
    - Cattle, Sheep

Sporadic Abortion

  1. Paenibacillus larvae
    - Honey bees

Amarican Foulbrood

30
Q

GENUS BACILLUS: PATHOGENICITY

SAPHROPHYTE BACILLI

A

= 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

31
Q

GENUS BACILLUS: PATHOGENICITY

Most imp bacillus bacteria for vet med

A
  1. B. Anthracis
  2. B. Subtilis
  3. B. Megaterium
  4. B.Cereus
32
Q

GENUS BACILLUS: PATHOGENICITY

Properties of B. ANTHRACIS

A

Flagella = NO

Capsule production in air = NO

Hemolysis = NO

Pathogenicity = YES

33
Q

GENUS BACILLUS: PATHOGENICITY

Properties of B. SUBTILIS

A

Flagella = YES

Capsule production in air = YES

Hemolysis = NO

Pathogenicity = NO

34
Q

GENUS BACILLUS: PATHOGENICITY

Properties of B. MEGATERIUM

A

Flagella = YES

Capsule production in air = YES

Hemolysis = NO

Pathogenicity = YES

35
Q

GENUS BACILLUS: PATHOGENICITY

Properties of B. CEREUS

A

Flagella = YES

Capsule production in air = YES

Hemolysis = YES

Pathogenicity = YES

36
Q

GENUS CLOSTRIDIUM - HABITAT

A

Soil

Mud

Water

Gut

37
Q

GENUS CLOSTRIDIUM - MORPHOLOGY

A

7-10 micro.m ROD

Flagella (exc. C.perfr)

SPORE (Terminal, subterminal, central)

=Help identification

No capsule (exc. C, perfr)

38
Q

GENUS CLOSTRIDIUM - RESISTANCE

A

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

39
Q

GENUS CLOSTRIDIUM - STAINING

A

GRAM +

40
Q

GENUS CLOSTRIDIUM - CULTURE

A

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

41
Q

GENUS CLOSTRIDIUM - BIOCHEMISTRY/ ANTIGENS

A

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

42
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

What are the different diseases caused by GENUS CLOSTRIDIUM

A

1. Gas gangrenic Diseases

- Malignant oedema (Cattle, Pig, Sheep)

- Black leg (Cattle, Sheep)

- Human gas gangrene

2. Enterotoxaemic Diseases

3. Intoxications

43
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

1. Gas Gangrenic Diseases

A

= 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

44
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

  1. Enterotoxaemic Diseases
A

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

45
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

  1. Intoxications
A

TETANUS = Rigid paralysis

C. Tetani

BOTULISM = FLACCID Paralysis

C. Botulinium

46
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. Septicum

A

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)

47
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

TOXIN TYPE A

A
  1. Necrotic Enteritis in Chickens
  2. Necrotizing enterocollitis in pigs
  3. Canine hemorrhagic enterocolitis
  4. Human gas gangrene, food infection
48
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

TOXIN TYPE B

A
  1. Lamb dysentery
  2. Haemorrhagic enteritis in calves and foals
49
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

TOXIN TYPE C

A

1. Necrotic Enteritis of Pigs

2. Struck in Sheeps

3. Necrotic enteritis in Chicken

50
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

TOXIN TYPE D

A

1. Pulpy kidney disease in sheep

2. Enterotoxaemia in calves, goats

51
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

TOXIN TYPE E

A

1. Haemorrhagic enteritis in calves

2. Enteritis in rabbits

52
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

All the bacterias

A
  1. C. Novyi
  2. C. Haemolyticum
  3. C. Histolyticum
  4. C. Chauvoei
  5. C. Perfringens
  6. C. Colinum
  7. C. Diffcile
  8. C. Tetani
  9. C. Botulinum
  10. C. Piliforme
  11. C. Spiroforme
53
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. NOVYI

A

- 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

54
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. HAEMOLYTICUM

A

Cattle, Sheep

BACILLARY HAEMOGLOBINUIA

55
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. HISTOLYTICUM

A

Whide spread

Can tollerate O2

Toxins: Alpha, Collagenase, Protease, elastase, haemolysin

Cattle, Pig, Sheep: MALIGNANT OEDEMA

56
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. CHAUVOEI

A

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)

57
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PERFRINGENS

A

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

58
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. COLINUM

A

Quail, Chicken, Turkey, Phesant:

ULCERATIVE ENTERITIS:

  • Intestinal Ulceration,
  • Hepatic Necrosis
59
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. DEFFICILE

A

Enterotoxin, Cytotoxin

Human

Pseudomembranaceus colitis

Foal

Hemorrhagic enterocolitis in newborn foals

Dog

Chronic Diarrhoea

60
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. TETANI

A

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

61
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. BOTULINUM

A

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)

62
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. PILIFORME

A

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

63
Q

GENUS CLOSTRIDIUM - PATHOGENICITY

C. SPIROFORME

A

Atypical COILED MORPHOLOGY

Rabbit: ENTERITIS

Pred factor:

Low fiber diet or AB Treatment

Iota-toxin production

Haemorrhagic Enteritis, Diarrhoea

FATAL WITHIN 48 HOURS

64
Q

Epsilon- and iota toxins are activated by

A

trypsin!

65
Q

Trypsin inactivates

A

Beta-toxin

66
Q

Differentiation of the most important Bacillus sp.

FLAGELLA

A

NOT B. anthracis

67
Q

Differentiation of the most important Bacillus sp.

CAPSULE (air)

A

ONLY

B. subtilis

B. megaterium

68
Q

Differentiation of the most important Bacillus sp.

HEMOLYSIS

A

ONLY

B. cereus

69
Q

Differentiation of the most important Bacillus sp.

PATHOGENICITY

A

ONLY

B.ANTHRACIS

70
Q

B. cereus show strong β-haemolysis

True or false

A

True

71
Q

What kind of clinical effect of tetanus

A

RIGID PARALYSIS

72
Q

What kind of clinical effect in Botulism

A

FLACCID Paralysis

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