Exercise 2 - 9 TABLES AND POINTERS

Four-quadrant streak plate method
identifier: colonies in the 4th quadrant

Identify method of isolation pure culture

Aseptic technique

Purpose of alcohol lamp

Nutrient agar

Culture medium used

Fourth Quadrant

which quadrant are colonies isolated

Serial Dilution Pour Plate Method
Identifier: there is label for serial dilution in the longer test tube

Identify method of isolation pure culture

Melted nutrient agar

Culture medium used

Ten-folds dilution

Two-folds dilution

Type of dilution used in the test

1:1000
1:60

Quantitative: Serial Dilution Pour Plate Method

Qualitative: Four-quadrant streak plate method

which is the quantitative and qualitative method for isolation of pure culture

37°Celsius for 24 hours

Incubation requirement for isolation of pure culture

Quebec colony counter

Principle: pressure sensing device

What equipment is used to quantitate the colonies in this test (include its principle)

Colony Forming Units/mL = colony count x reciprocal of dilution

Too few to count formulae (you are counting all the colony in the petri dish)

Average number of colonies in five squares x 62.5 x reciprocal of dilution=Colony Forming Units/mL

Too numerous to count formulae

Serial Dilution Pour Plate Method

Identify method of isolation pure culture

Too many to count

First three dilutions interpretation
1:10
1:100

1:1 000

Too few to count

last three dilutions interpretation
1:10 000

1:100 000

1:1 000 000

As the dilution increases the colony count decreases

As the dilution _ the colony count _

62.5

diameter of petri dish

Disinfection

bacteriostatic; inhibition

Sterilization

bacteriocidal; killing

• 80°Celsius

• Boiling: 100°Celsius

• Autoclave: 121°Celsius

• Hot air: 180°Celsius

temperature of physical agents
_

boiling
autoclave

hot air

Endospores; Resists disinfection

What is responsible for the characteristic result of the organism of letter A to this test and its action

Nutrient broth

medium used

• 80°Celsius

• Boiling: 100°Celsius

What are the physical agents of disinfection

• Autoclave: 121°Celsius

• Hot air: 180°Celsius

What are the physical agents of sterilization

Physical agents of disinfection

• 80°Celsius

• Boiling: 100°Celsius

Physical agents of sterilization

• Autoclave: 121°Celsius

• Hot air: 180°Celsius

Turbidity

Only evidence of bacterial growth of this test

• Turbidity

• change of pH in the medium

• presence of Hydrogen gas in the Durham’s fermentation tube
  
  (if any of these are indicators are positive there is growth of bacteria)

3 evidence of bacterial growth

(disinfectant) killed the bacteria in (time)

How do you interpret the result

answer the time only (ex. 30 mins)

Contact time of the disinfectant to kill the organism

To trap Hydrogen gas

Purpose of Durham’s fermentation tube in the culture medium

• Andrade’s indicator

• peach

• pink

pH indicator in the culture medium or the responsible component of the change in color of the medium

• name

• original color

• change in color if there is change in pH

Lactose broth with Durham’s fermentation tube

culture medium

• Coliform

• Gram-negative non-sporogenous lactose fermenter organisms

• non-sporogenous: there is no endospores

type organisms that produce HYDROGEN gas

• name

• description

Antimicrobial susceptibility test

identify test

Kirby-Bauer method

identify method used in the test

• 0.5 McFarland standard

• Composition

  • Volume: 99.5 ml of 1% sulfuric acid; 0.5 ml of 1.175% barium chloride

  • Concentration:1.5 x 10⁸ CFU/ml

bacterial suspension used in this test is compared to the turbidity of what material and its composition

Mueller-Hinton agar

culture medium used in the test

• Beef Extract – 2.0 g/L

• Acid Hydrolysate of Casein (Peptone) – 17.5 g/L

• Starch – 1.5 g/L

• Agar – 17.0 g/L

• Distilled Water – 1,000 mL

• Magnesium Ions (Mg²⁺) – 20–25 mg/L

• Calcium Ions (Ca²⁺) – 50 mg/L

• Final pH – 7.2-7.4 at 25°C

• trace amounts of thymidine, usually less than 1 mg/L

Supplements in Mueller Hinton agar

Gram’s Staining/ Hucker’s method

identify method

Staphylococci

identify organism investigated

• pigment production

• Presumptive test

identify test and protocol

• Catalase test

• Presumptive test

identify test and protocol

• Coagulase test

• Confirmatory test

identify test and protocol

• Slant portion; aerobic condition

• Butt portion; anaerobic condition

parts in the tube and its conditions

simple streaking

streaking used

• Oxygen

element responsible for the positive result

complete hemolysis (BA turns clear color of TSA is seen)

beta hemolysis (and its description)

Partial hemolysis (greenish brown)

alpha hemolysis (and its description)

no hemolysis (no change in culture media)

gamma hemolysis (and its description)

VIRULENCE

Ability of the microorganism to cause infection or disease.

STRUCTURES & COMPOSITION

Things or substances present within the organism which they utilize for them to cause infection to human host

irregular shaped circles

GRANULAR

inside the body

INCLUSION

ACCUMULATION OF METAPHOSPHATES

composition of the bacterial structure, granular inclusion body

NEUTROPHILS, EOSINOPHILS, & MONOCYTES

3 types of phagocytes

Metachromasia

A special property of granular inclusion bodies/granules where it appears purple when stained with methylene BLUE

metachromatic granules

GIB are also called

diphtheria and leprae

causative agent: Corynebacterium diphtheriae

Babes-Ernst granules
Chinese letter appearance

specific name of the metachromatic granules of Corynebacterium diphtheriae and its morphology

fibrous protein

structure of composition

flagellin

composition

Tannic acid

specific reagent necessary for the demonstration of the flagella

MORDANT

what reagent is necessary for the demo of flagella

1. NO OF FLAGELLA PRESENT IN ORGANISM

2. LOCATION OF THE FLAGELLA IN THE BODY OF THE ORGANISM

criteria of MESSEA’s classification

Monotrichous - one flagella

Lophotrichous - several flagella in a tuft(bundle)

Amphitrichous - one flagella each side

Peritrichous - flagella all over the body

Atrichous - no flagella

Monotrichous

Lophotrichous

Amphitrichous

Peritrichous

Atrichous

Pseudomonas aeruginosa - monotrichous
Salmonella typhi - peritrichous

Representative Organisms and their flagella characteristics

Drumstick appearance

Terminal spores description

scanty amount

slime layer amount

copious amount

capsule amount

NEGROSIN - detects capsule that produces semi-opaque background

QUELLUNG REACTION - ab-ag (antiserum) swells the capsule

TESTS DONE for capsules and description

COMPLEMENT SYSTEM

second line and highest form of defense against any invading antigen

CELL-LYSIS OR CELL DEATH

end action of the complement system when the immune system is activated

SIMPLE STAINING

1 dye only
morphology of the bacteria in films

direct staining

DIFFERENTIAL STAINING

2 or more dyes

principle of dfs: bacteria differ chemically and physically and may react differently to a given staining procedure

principle of dfs: bacteria differ _ and _ and may react _ to a given staining procedure

1 uL

volume a wire loop can hold

• Primary Staining

  • Crystal violet(HEXAMETHYL-PARA-ROSANILINE CHLORIDE)

  • alternative: Gentian violet

  • 1 min

• Mordant

  • Gram’s iodine (brown-colored stain)

  • 1 min

• Decolorization (crucial step)

  • Acetone alcohol

  • 3-5 seconds

• Counter Staining

  • Safranin (red-colored stain)

  • 45 secs

DFS steps, stain used, time

THICK PEPTIDOGLYCAN LAYER WITH TEICHOIC ACID (amino acids and disaccharides)

Gram-positive organisms’ cell wall

THIN PEPTIDOGLYCAN LAYER WITH LIPOPOLYSACCHARIDES (lipids and polysaccharides)

Gram-negative organisms’ cell wall

simultaneously kills the microorganism

allows the smear to adhere

heat fixing

preserves morphology

air drying

MORDANT

intensifies the color of the primary stain to the cell wall

CRYSTAL VIOLET-IODINE COMPLEX

iodine molecule forms an attachment with the crystal violet molecule making…

may appear gram negative

gram positive organism and the crystal violet was rinsed vigorously

PURE CULTURE

only one species of bacteria

NUTRIENT AGAR

simple culture medium used for the isolation of non fastidious organisms

Pepton Digest of animal tissue: 5 grams per liter

Beef extract 1.5 grams per liter

NaCl 5 grams per liter

Agar 15 grams per liter

Sterile water

NUTRIENT AGAR composition

• Used to quantitate colonies

• Count the surface and subsurface colonies

• pressure-sensing device

• beeping sound

Quebec Counter

• use

• counts the what colonies

• principle

• indicator

psychrophiles

incubation is unsuitable for

principle of Four quadrant streak plate method: by streaking, dilution gradient is established on the surface of the plate as cells are deposited on the agar surface

principle of Four quadrant streak plate method: by _ , dilution _ is established on the _ of the plate as _ are deposited on the agar _

appearance

color

edge

elevation

shape

size

Criteria used to characterize bacterial growth

pasteur pipette

joseph lister

used for dispensing to the petri dish in serial dilution pour plate method invented by

• 0.85 g; 100mL

• isotonic

• preserves morphology

normal saline solution

• composition

• type of solution

• action

STERILIZATION

All forms of microbial life including bacterial endospores are killed.

DISINFECTION

inhibits pathogenic organisms but not necessarily all microorganisms or endospores are destroyed.