Ceu - Microbio 1 Lab

8/6/2019 Ceu - Microbio 1 Lab

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nucleoid

DNA in the bacterial cell is generally confined to

this central region. Though it isn't bounded by a

membrane, it is visibly distinct (by transmissionmicroscopy) from the rest of the cell interior.

ribosomes

Ribosomes give the cytoplasm of bacteria a

granular appearance in electron micrographs.

Though smaller than the ribosomes in eukaryotic

cells, these inclusions have a similar function in

translating the genetic message in messengerRNA into the production of peptide sequences

(proteins).

storage granules

(not shown) Nutrients and reserves may be

stored in the cytoplasm in the form of glycogen,

lipids, polyphosphate, or in some cases, sulfur or

nitrogen.

endospore

(not shown) Some bacteria, like Clostridium

botulinum, form spores that are highly resistant to

drought, high temperature and other

environmental hazards. Once the hazard is

removed, the spore germinates to create a new

population.

Internal Structure: Bacteria have a very simple internal structure, and no membrane-bound

organelles.


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Beginning from the outermost structure and moving inward, bacteria have some or all of the following structures

:

capsule

This layer of polysaccharide (sometimes proteins)

protects the bacterial cell and is often associated

with pathogenic bacteria because it serves as abarrier against phagocytosis by white blood cells.

outer membrane

(not shown) This lipid bilayer is found in Gram

negative bacteria and is the source of

lipopolysaccharide (LPS) in these bacteria. LPS is

toxic and turns on the immune system of , but not in

Gram positive bacteria.

cell wall

Composed of peptidoglycan (polysaccharides +protein), the cell wall maintains the overall shape of

a bacterial cell. The three primary shapes in

bacteria are coccus (spherical), bacillus (rod-

shaped) and spirillum (spiral). Mycoplasma are

bacteria that have no cell wall and therefore haveno definite shape.

periplasmic space

(not shown) This cellular compartment is found onlyin those bacteria that have both an outer membrane

and plasma membrane (e.g. Gram negative

bacteria). In the space are enzymes and other

proteins that help digest and move nutrients into the

cell.

plasma membrane

This is a lipid bilayer much like the cytoplasmic

(plasma) membrane of other cells. There arenumerous proteins moving within or upon this layer


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appendages

pili

These are hollow, hairlike structures made

of protein allow bacteria to attach to other

cells. A specialized pilus, the sex pilus,

allows the transfer from one bacterial cell

to another. Pili (sing., pilus) are also called

fimbriae (sing., fimbria).

flagella

The purpose of flagella (sing., flagellum) ismotility. Flagella are long appendages

which rotate by means of a "motor" located

just under the cytoplasmic membrane.

Bacteria may have one, a few, or many

flagella in different positions on the cell.


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Psychrophilic

Mesophilic

Thermophilic

Thermoduric

Halophilic

Capnophilic


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1. Lag Phase adaptation of bacteria to its new

environment

2. Log/Exponential phase bacterial division at

constant rate

3. Stationary Phase decrease in bacterial growthrate

4. Death Phase/Phase of Decline complete

cessation or stoppage of bacterial multiplication


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Methods of STERILIZATION

1. MOIST HEAT

1.1 boiling 100 deg C for15-30 mins

1.2 fractional sterilization

a) Tyndallization- flowing steam 30 mins for 3

days at 100 deg C

b) Inspissation- 75-80 degC 2 hrs for 3 days

c) Pasteurization 60 degC for 30 mins

1.3 Autoclaving 121 degC 15-30 mins at 15 lbs psi

2. DRY HEAT

2.1

oven -160

-1

80

deg C1-2 hrs for glasswares

2.2 flame

2.3 incineration 300-400 degC


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3. FILTRATION

3.1 asbestos filter ( Seitz )

3.2 membrane filter ( millipore filter0.22 um )4. Lyophilization

5. Ultracentrifugation

6. ETHYLENE OXIDE GAS ( cold sterilization )

7. DISINFECTANTS AND ANTISPETICS

5.1 disinfectant for thermometers, surgical instruments

i.e. hypochlorite, quaternary ammoniums like zephiran

5.2 antiseptic

i.e. alcohol, tincture iodine/alcoholic iodine, iodophor

* Bactericidal and Bacteriostatic


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Ways to study Microorganisms under the

Microscope

A) LIVING STATE

1. Wet Mount maybe carried out by:

1.1 placing a loopful of liquid specimen on a slide andcovering it with a coverslip

1.2 emulsifying non-liquid specimen using NSS on a

slide then covering it with a coverglass

2. Hanging Drop almost the same as the wet mount but loopful oforganisms are placed on a coverslip. The coverslip is

then inverted over a concave portion of a slide toprovide the hanging drop. Essential for demonstratingmotility

B) FIXED STATE - carried out by preparing a smear, allowing it to dry,

fixing and staining.


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PROCEDURE OF HANGING DROP TECHNIQUE

If concavity slide is to be used:1. Place a loopful of organism at the center of a coverslip

2. Invert coverslip over the concave portion of a slide.

3. Examine under the microscope , LPO and HPO

If Ordinary slide is to be used:1. Spread a small amount of petroleum on a slide to make a

hollow depression.

2. Place a loopful og organism on a coverslip

3. Invert the slide over the coverslip in such a way that thecenter of depression lies over the drop

4. Invert the slide now so that the drop to be examined

hangs from the bottom of the coverslip

5. Examine under the microscope


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Procedure inPreparing a Bacterial Smear

1. Sterilize wire loop

2. Using a sterilize loop, pick a small colony and

emulsify in a drop of distilled water. If liquid

organism is to be used, place it directly on aslide ( no need to emulsify )

3. Allow it to dry

4. Fix smear by passing smear over the flame.5. Stain the smear with the desired staining

process


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I. STUDY OF MORPHOLOGY

a) size

b) shapec) arrangement

d) motility

1. motile

2. non-motile

* Brownian Movement

e) staining characteristics


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V - crystal violet

I - IODINE

A - 9 5% alcohol or mixture

of alcohol and acetone

S - SAFRANIN

Gram Positive = PURPLE

Gram Negative= RED


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All cocci are gram (+) except : NEISSERIA ,VEILONELLA & BRANHAMELLA

All bacilli are gram (-) except: MYCOBACTERIUM,CORYNEBACTERIUM, CLOSTRIDIUM,

BACILLUS, ERYSIPELOTHRIX, LISTERIA,LACTOBACILLUS

Higher forms of organisms likeACTINOMYCES ,STREPTOMYCES, yeast and molds are gram

(+)


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Procedure of Gram Staining

1. Prepare a bacterial smear

2. Overlay smear with Crystal violet for 30 sec 1 min3. Rinse with distilled water, tapping off excess

4. Flood smear with iodine for1 min

5. Rinse with distilled water , tapping off excess

6. Add acetone or ethyl alcohol drop by drop until noviolet color appears in rinse . This requires less than

10 secs

7. Rinse with distilled water immediately

8. Flood smear with safranin for 30 secs9. Rinse with distilled water and allow slide to drain

10. Blot dry and examine


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ACID FAST STAINING

ACID FAST ORGANISM these are organismsthat are very hard to stain but once stained they

are difficult to decolorize due to MYCOLIC

ACID / HYDROXYMETHOXY ACID thatenvelopes the bacteria

Rule : All bacteria are Non-Acid Fast except :Mycobacterium, Slightly Acid Fast is Nocardia


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1. Steaming process

2.Increasing concentrationof phenol blue and basicfuchsin

3.Prolonging contact of

stain with the material4.Addition of wetting

agents ( tergitol ) to thestain solution


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1. ZIEHL NEELSEN

C carbol fuchsin

A acid alcohol

M methylene blue

result : Acid Fast = RED

Non-Acid Fast = BLUE


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2. Kinyouns/ Cold Method

C carbol fuchsin

A acid alcohol

M malachite green

* no steaming process

Result : Acid Fast = RED

Non-Acid Fast = GREEN


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3. Pappenheims differentiates M.

smegmatis from M. tuberculosis

4. Baumgartens differentiates M.

tuberculosis from M. leprae


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Procedure of Acid Fast Staining ( Ziehl Neelsen )

1. Prepare a bacterial smear

2. Flood smear with carbol fuchsin

3. Gently steam over flame for 3-5 minutes( do not boil )

4. Wash off excess stain with water

5. Decolorize for15-20 secs with acid alcohol

6. Counterstain with methylene blue for1 minute

7. Wash with water and allow it to dry and examine


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Procedure of Negative Staining

1. Place a drop of nigrosin/india ink on a slide

2. Add a loopful of culture on the drop

3. Using the edge of another slide, spread the drop out

4. Allow smear to dry but do not fix

5. Examine

Result: bacteria appears colorless against a grayishblack background


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II. STUDY OF CULTURAL CHARACTERISTICS* Culture media

CLASSIFICATION OF CULTURE MEDIA

a) According to Physical State/Consistency

1. Liquid2. semi-solid with 0.5 1.5 % agar

3. solid with 1.5 3.0% agar

b) According to how media is dispensed

1. plated

2. tubedc) According to Use

1. general isolation media

i.e. Nutrient Broth , BHI, TSB

2. Enrichment media

i.e. Selenite broth, GN broth, tetrathionate broth


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3. Selective media

gentian violetmethylene blue inhibits gram

Na deoxycholate & other bile salts positive organisms

Vancomycin and penicillin

potassium tellurite

sodium azide inhibits gram

alcohol negative organisms

chloral hydrate

PEA ( phenylethyl alcohol alcohol agar )- allows growth of

gram positive cocci while inhibiting growth of gram negative


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4. differential media i.e. BAP

5. Selective and differential media

i.e. Mac conkey, EMB, XLD

6. Special media

i.e. Fletchers Leptospira

Bordet-Gengou B. pertussis


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1. Culture the bacteria

2. Identify the cultured bacteria

Methods of identification:

a) morphological

b) biochemical tests

c) serological testsd) use of nwly discovered

techniques like DNA

hybridization

3. Test the susceptibility of bacteria

to antimicrobial agents


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1. Liquid media

2. Slanted media

3. Butt media

4. Butt/slant media

5. Plated media

1. radial streak

2. overlap streaking

3. multiple streaking

INOCULATION TECHNIQUE IN THE CULTIVATION OF

BACTERIA

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Ceu - Microbio 1 Lab