III: Bacterial Identification

1. S - -Size

2. S - Shape

3. A - Arrangement

4. M - Motility

5. S - Staining characs

Bacteria can be identified based on their morphology and appearance such a s

STAINING

the process of artificially coloring the organism with dye/ stains

STAINING

Its purpose is to observe and appreciate the appearance of bacteria

STAINING

Its purpose is to differentiate one organism from the other

STAINING

Its purpose is to reveal the chemical nature of bacteria

1. Simple / Direct

2. Differential

3. Indirect / Relief / Negative

4. Special Staining

Staining Techniques

Simple/ Direct

one dye; all organisms have the same color

Differential

two dyes; (GS and AFS)

Indirect/Relief/ Negative

Capsule

a. India Ink Test/ Borris Method

b. Nigrosin Method

2 types of Indirect/Relief/ Negative

Special staining

to demonstrate special features of the cell

1. Muir

2. Anthony's

3. tyle

4. Hiss

5. Welch's Grins

Special Staining

Capsule

1. Dorner's Schaeffer-Fulton

2. Wirtz and Conklin

3. Heat and Acetic Acid Method

Special Staining

Spores

1. Gray's

2. Fisher and Conn

3. Loeffler's

4. Leifson

5. Caesares

6. Gil

7. Van Ermenger

Special Staining

Flagella

1. Feulgen

2. Acridine orange

Special Staining

Nucleic Acid

Wayson

Special Staining

Polar Bodies

1. Levaditi

2. Fontana

3. Warthin- Starry

Special Staining

Spirochetes

1. Gimenez

2. Macchiavelo

3. Giemsa

Special Staining

Rickettsia

1. Neisser

2. Albert's

3. Ljubinsky

4. Lindergan

5. Ponder

Special Staining

Metachromatic Granules

Hans Christian Gram

GRAM STAINING is Developed by Danish bacteriologist - in 1884

teichoic acid

Gram staining Stains the - present on the cell walls of gram-positive

T

Gram staining is Not applicable to organisms that exist almost exclusively within host cells (Chlamydia) those that lack cell walls, and those of insufficient dimension to be resolved by light microscopy (Spirochetes) (t/f)

1. N - Neisseria

2. V - Veilonella

3. M - Moraxella

Al cocci are gram (+) except

1. M - Mycobacteria

2. C- Corynebacteria

3. C - Clostridia

4. B - Bacillus

5. E - Erysiphilotrix

6. L - Lactobacillus

7. L - Listeria

8. N - Nocardia

9. A - Actinomyces

Al bacilli are gram (-) except

gram -

All spiral organisms are reported as

gram +

Yeasts are reported as

Chlamydia/ Rickettsia

Cannot be gram-stained

- intracellular

Mycoplasma/ Ureaplasma

Cannot be gram-stained

- no cell wall

Spirochetes

Cannot be gram-stained

-- can't be resolved by LM

• Legionella & Spirochete

Cannot be gram-stained

-- silver impregnation technique is most useful

I. Living state (unstained)

I. Fixed State (Stained)

Methods ofStudying Microorganisms

1. Wet mount Preparation Specimen mixed with NSS

2. Hanging drop preparation-

be uses concave slide; can used to demonstrate the motility of the organism

Steps in Living state (unstained)

1. Bacterial Smear Preparation

2. Fixation- Using heat or 70- 95 % alcohol; 95% methanol

3.Staining

Steps in Fixed state (stained)

heat or 70- 95 % alcohol; 95% methanol

Fixation is done using

Hanging drop preparation

uses concave slide; can used to demonstrate the motility of the organism

Steps in Gram Staining

G (+)becomesG (-)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

Over-decolorization

G (+)becomesG (-)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

Old, dying

G (+)becomesG (-)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

Use of acidic iodine as a mordant

G (+)becomesG (-)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

Penicillin (loss of cell wall integrity)

G (+)becomesG (-)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

lodine/ mordant was omitted

G(-) becomes G(+)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

under-decolorization

G(-) becomes G(+)

ERRORS IN GRAM STAINING

Identify if G (+)becomesG (-) or G(-) becomes G(+)

Thick smear

VAIAS

MODIFICATIONS OF GRAM STAINING

Hucker’s Modification Steps

VISES

MODIFICATIONS OF GRAM STAINING

Burke’s Modification Steps

Burke’s Modification

stain that shows the metachromatic granules

G (+)

Identify if G (+) or G (-)

Cell wall

THICK cell wall of composed peptidoglycan; TEICHOIC ACIDS may be present

G (-)

Identify if G (+) or G (-)

Cell wall

The outer membrane composed of lipids, protein, lipopolysaccharides, and inner thin peptidoglycan

G (+)

Identify if G (+) or G (-)

Shape

Spherical, rods, or filaments

G (-)

Identify if G (+) or G (-)

Shape

Spherical, ovals, straight, curve, rods, helical etc

G (+)

Identify if G (+) or G (-)

Teichoic Acid

PRESENT

G (-)

Identify if G (+) or G (-)

Teichoic Acid

Absent

G (+)

Identify if G (+) or G (-)

Endospores

PRESENT in some groups

G (-)

Identify if G (+) or G (-)

Endospores

Absent

G (+)

Identify if G (+) or G (-)

Periplasmic Space

ABSENT

G (-)

Identify if G (+) or G (-)

Periplasmic Space

Present

G (+)

Identify if G (+) or G (-)

Flagellar structure

2 rings in basal body

G (-)

Identify if G (+) or G (-)

Flagellar structure

4 rings in basal body

G (+)

Identify if G (+) or G (-)

Resistance to Physical Disruption

HIGH

G (-)

Identify if G (+) or G (-)

Resistance to Physical Disruption

LOW

G (+)

Identify if G (+) or G (-)

Inhibition by Basic Dyes

HIGH

G (-)

Identify if G (+) or G (-)

Inhibition by Basic Dyes

LOW

mycolic acid

ACID-FAST STAINING

Stains the - in the cell wall of bacteria

F

ACID-FAST STAINING

Organisms with Mycolic acid/ hydroxyl methoxy acid in the cell wall are easy to gram-stain (t/f)

1. Mycobacteria

2. Nocardia (partially acid-fast)

3. Cryptosporidium

4. Cyclospora

ACID-FAST STAINING

Acid-fast organism:

1. Steaming

2. Addition of wetting agent (tergitol) prior to the stain solution

3. Increasing concentration of phenol (accentuator) and basic fuchsin

4. Prolonging contact of stain with the material

Ways to Facilitate Acid Fast Organisms

ACID-FAST STAINING

Ziehl-Neelsen Stain Steps

ACID-FAST STAINING

Kinyoun's Method

*1% H2SO4, 70% Ethanol

ACID-FAST STAINING

Modified Kinyoun's Method for Partially/ Weakly acid- Fast

Pappenheim's Method

Other Modifications of ACID-FAST STAINING

used to differentiate Mycobacterium smegmantis (neg) from Mycobacterium tuberculosis (pos)

Fite-Faracos/Auramine-Rhodamine

Other Modifications of ACID-FAST STAINING

used to differentiate M. leprae (red-pos) from M. tuberculosis (blue- neg)

Culture

microorganisms in specimens can be cultivate and grown in artificial media

Culture medium

contains nutritional requirements needed for bacterial growth

1. Pure Culture

2. Mixed Culture

3. Stock Culture

TYPES OF CULTURE

Pure Culture

one species

Mixed Culture

- more than one species

Stock Culture

- for ATCC (known stain bacteria)

1 Weigh

2. Dissolve in deionized or distilled water

3. Sterilize

4. Dispense

WDSD

STEPS IN PREPARING CULTURE MEDIA

Plated (Petri Dish)

1. Weigh

2. Dissolve in deionized or distilled water

3. Dispense

4 . Sterilize

WDDS

STEPS IN PREPARING CULTURE MEDIA

Tube (Test Tube)

A. According to the Physical State

1. Liquid

2. Semi-solid

3. Solid

4. Biphasic

B. According to Composition (SNT)

1. Synthetic/ Defined

2. Non-synthetic/ Complex

3. Tx cells

C. According to Purpose (DTESSS)

1. Simple /General Isolation

2. Enriched

3. Enrichment

4. Selective

5. Differential

6. Transport

TYPES OF CULTURE MEDIA

0 % or none

Amount of solidifying agent of Liquid

0.5-1%

Amount of solidifying agent of Semi-solid

2 - 3%

Amount of solidifying agent of Solid

Solid & liquid

Amount of solidifying agent of Biphasic

Liquid

Identify if what type of culture media according to physical state is the examples

1. Nutrient Broth

2. Brain Heart Infusion

3. Alkaline Peptone Water

4. Thioglycolate

Semi-solid

Identify if what type of culture media according to physical state is the examples

1. S I M

2. Gelatin Media

Solid

Identify if what type of culture media according to physical state is the examples

Liquifiable:

Liquid-> Solid-> Liquid

Ex. SSA, BAP, MAC

Non-liquifiable:

Will no longer liquefy when heated again

Ex. Rice medium

Bipahasic

Identify if what type of culture media according to physical state is the examples

1. HBT- Human Blood Bilayer Tween (G. vaginalis)

2. Castanedas- Brucella

Synthetic/ Defined

-All components are KNOWN to the user

-for research

BG-II Cyanobacteria

eg Synthetic/ Defined

Non-synthetic / Complex

-composed of some UNKNOWN

-substance (peptone, meat broth)

-very useful for isolation of bacteria

1. Nutrient Broth

2. TSB

3. MacConkey Agar

eg Non-synthetic / Complex

Tx cells

-uses living cell

- we look for CYTOPATHIC EFFECT

1. HeLA cells

2. A549

3. McCoy Cells

4. Vero Cells

5. Hep2 Cells

eg Tx cells

Simple /General Isolation

It is known as supportive media, supports the growth of most non-fastidious bacteria

Simple /General Isolation

No growth advantage is given to any group of bacteria

1. Nutrient agar

2. trypticase soy agar

3. nutrient broth

eg Simple /General Isolation

Enriched

Used for growing FASTIDIOUS ORGANISMS

Enriched

For general isolation with added nutrients like blood, serum, peptone, and vitamins

1. BAP

2. CAP

eg of Enriched