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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
GENUS BACILLUS: PATHOGENICITY Horses
Subacute **ANTHRAX** with **localized oedema**, **septicemia** with **collic** and **enteritis** sometimes occur
26
GENUS BACILLUS: PATHOGENICITY Humans
**Skin**, **Pulmonary** and **intestinal** form of **ANTHRAX**
27
GENUS BACILLUS: PATHOGENICITY CARNIVORES
Comparatively resistant
28
GENUS BACILLUS: PATHOGENICITY BIRDS
**Completely resistant**
29
GENUS BACILLUS: PATHOGENICITY Types of bacteria
1. **B. Cereus** - Cattle: Mastitis (Rare) - Humans: Food Poisoning (GI infections) Eye infections 2. **B. Licheniformis** - Cattle, Sheep Sporadic Abortion 3. **Paenibacillus larvae** - Honey bees Amarican Foulbrood
30
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
31
GENUS BACILLUS: PATHOGENICITY Most imp bacillus bacteria for vet med
1. B. Anthracis 2. B. Subtilis 3. B. Megaterium 4. B.Cereus
32
GENUS BACILLUS: PATHOGENICITY Properties of B. ANTHRACIS
Flagella = NO Capsule production in air = NO Hemolysis = NO **Pathogenicity = YES**
33
GENUS BACILLUS: PATHOGENICITY Properties of **B. SUBTILIS**
**Flagella = YES** **Capsule production in air = YES** Hemolysis = NO Pathogenicity = NO
34
GENUS BACILLUS: PATHOGENICITY Properties of **B. MEGATERIUM**
**Flagella = YES** **Capsule production in air = YES** Hemolysis = NO Pathogenicity = YES
35
GENUS BACILLUS: PATHOGENICITY Properties of **B. CEREUS**
**Flagella = YES** Capsule production in air = YES **Hemolysis = YES** Pathogenicity = YES
36
GENUS CLOSTRIDIUM - HABITAT
Soil Mud Water Gut
37
GENUS CLOSTRIDIUM - MORPHOLOGY
**7-10** micro.m **ROD** **Flagella** (exc. C.perfr) **SPORE** (Terminal, subterminal, central) =Help identification **No capsule** (exc. C, perfr)
38
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**
39
GENUS CLOSTRIDIUM - STAINING
**GRAM +**
40
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**
41
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**
42
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**
43
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**
44
GENUS CLOSTRIDIUM - PATHOGENICITY 2. 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**
45
GENUS CLOSTRIDIUM - PATHOGENICITY 3. Intoxications
TETANUS = **Rigid** paralysis **C. Tetani** BOTULISM = **FLACCID** Paralysis **C. Botulinium**
46
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)
47
GENUS CLOSTRIDIUM - PATHOGENICITY ## Footnote **C. PERFRINGENS** **TOXIN TYPE A**
1. **Necrotic Enteritis** in Chickens 2. **Necrotizing enterocollitis** in pigs 3. Canine **hemorrhagic enterocolitis** 4. **Human gas gangrene**, food infection
48
GENUS CLOSTRIDIUM - PATHOGENICITY **C. PERFRINGENS** **TOXIN TYPE B**
1. **Lamb dysentery** 2. **Haemorrhagic enteritis** in calves and foals
49
GENUS CLOSTRIDIUM - PATHOGENICITY **C. PERFRINGENS** **TOXIN TYPE C**
**1. Necrotic Enteritis of Pigs** **2. Struck i**n Sheeps **3. Necrotic enteritis in Chicken**
50
GENUS CLOSTRIDIUM - PATHOGENICITY **C. PERFRINGENS** **TOXIN TYPE D**
**1. Pulpy kidney disease** in sheep **2. Enterotoxaemia** in calves, goats
51
GENUS CLOSTRIDIUM - PATHOGENICITY **C. PERFRINGENS** **TOXIN TYPE E**
**1. Haemorrhagic enteritis** in calves **2. Enteritis** in rabbits
52
GENUS CLOSTRIDIUM - PATHOGENICITY All the bacterias
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
GENUS CLOSTRIDIUM - PATHOGENICITY ## Footnote **C. NOVYI**
**- Toxins: Alpha, lipase, lecinase, DN-ase,** **- CollagenaseTypes**: **A, B, C** (**Non-pathogenic, no toxin**s) **1. Gas gangrene:** **Cl. Novyi A/B** **2. Sheep, Cattle: Infectious necrotic hepatitis:** **Cl. Novyi B**
54
GENUS CLOSTRIDIUM - PATHOGENICITY **C. HAEMOLYTICUM**
Cattle, Sheep **BACILLARY HAEMOGLOBINUIA**
55
GENUS CLOSTRIDIUM - PATHOGENICITY **C. HISTOLYTICUM**
Whide spread **Can tollerate O2** Toxins: Alpha, Collagenase, Protease, elastase, haemolysin Cattle, Pig, Sheep: **MALIGNANT OEDEMA**
56
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)
57
GENUS CLOSTRIDIUM - PATHOGENICITY **C. PERFRINGENS**
Optimal Temperature **40-45 degrees,** N**o Flagella, have Capsule** **Toxins:** Main: (alpha, beta, epsilon, iota - Activated by **TRYPSIN**) Auxiliary: (Gamma, Delta, Theta, Kappa, Lambda, Mu) - **Neutralization**
58
GENUS CLOSTRIDIUM - PATHOGENICITY **C. COLINUM**
Quail, Chicken, Turkey, Phesant: **ULCERATIVE ENTERITIS:** - Intestinal Ulceration, - Hepatic Necrosis
59
GENUS CLOSTRIDIUM - PATHOGENICITY C. DEFFICILE
**Enterotoxin, Cytotoxin** Human **Pseudomembranaceus colitis** Foal **Hemorrhagic enterocolitis in newborn foals** Dog **Chronic Diarrhoea**
60
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**
61
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)**
62
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**
63
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**
64
**Epsilon- and iota toxins are activated by**
**trypsin!**
65
Trypsin inactivates
Beta-toxin
66
Differentiation of the most important Bacillus sp. FLAGELLA
**NOT B. anthracis**
67
Differentiation of the most important Bacillus sp. **CAPSULE (air)**
**ONLY** **B. subtilis** **B. megaterium**
68
Differentiation of the most important Bacillus sp. **HEMOLYSIS**
## Footnote **ONLY** **B. cereus**
69
Differentiation of the most important Bacillus sp. **PATHOGENICITY**
**ONLY** **B.ANTHRACIS**
70
**B. cereus** show strong **β-haemolysis** True or false
**True**
71
What kind of clinical effect of tetanus
RIGID PARALYSIS
72
What kind of clinical effect in Botulism
FLACCID Paralysis

Bacto practical (9 decks)

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