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

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 transmission microscopy) from the rest of the cell interior. 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 messenger RNA into the production of peptide sequences (proteins). (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. (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.

nucleoid

ribosomes

storage granules

endospore

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 a barrier against phagocytosis by white blood cells. (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. 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 (rodshaped) and spirillum (spiral). Mycoplasma are bacteria that have no cell wall and therefore have no definite shape. (not shown) This cellular compartment is found only in 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. This is a lipid bilayer much like the cytoplasmic (plasma) membrane of other cells. There are numerous proteins moving within or upon this layer that are primarily responsible for transport of ions,

outer membrane

cell wall

periplasmic space

plasma membrane

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) is motility. 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.

Obligate aerobe Obligate anaerobe Facultative aerobe/facultative anaerobe Microaerophilic Aerotolerant

Autotrophic Heterotrophic

Psychrophilic Mesophilic Thermophilic Thermoduric

Halophilic

Capnophilic

1. Lag Phase ² adaptation of bacteria to its new environment 2. Log/Exponential phase ² bacterial division at constant rate 3. Stationary Phase ² decrease in bacterial growth rate 4. Death Phase/Phase of Decline ² complete cessation or stoppage of bacterial multiplication

Methods of STERILIZATION
1. MOIST HEAT 1.1 boiling ± 100 deg C for 15-30 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 DRY HEAT 2.1 oven - 160-180 deg C 1-2 hrs for glasswares 2.2 flame 2.3 incineration ± 300-400 degC

2.

3.

4. 5. 6. 7.

FILTRATION 3.1 asbestos filter ( Seitz ) 3.2 membrane filter ( millipore filter 0.22 um ) Lyophilization Ultracentrifugation ETHYLENE OXIDE GAS ( cold sterilization ) 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

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 and covering 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 of organisms are placed on a coverslip. The coverslip is then inverted over a concave portion of a slide to provide the hanging drop. Essential for demonstrating motility B) FIXED STATE carried out by preparing a smear, allowing it to dry, fixing and staining.

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 the center 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

Procedure in Preparing 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 a slide ( 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

I.

STUDY OF MORPHOLOGY a) size b) shape c) arrangement d) motility 1. motile 2. non-motile * Brownian Movement e) staining characteristics

A) B)

Purpose Staining Techniques 1. Simple Staining 2. Indirect/Relief/ Negative 3. Special Staining 3.1 capsular stains ( Hiss, Anthony¶s ) 3.2 Spore stains ( Dorner¶s , Schaeffer and Fulton, Wirtz conklin ) 3.3 flagellar stain ( Gray¶s, Fisher & Conn, Leifson ) 3.4 metachromatic granules ( Albert¶s, Neisser, Ljubinsky, Ponder, Methylene Blue ) 4. Differential Staining

V - crystal violet I - IODINE A - 95% alcohol or mixture of alcohol and acetone S - SAFRANIN Gram Positive = Gram Negative= PURPLE RED

All cocci are gram (+) except : NEISSERIA , VEILONELLA & BRANHAMELLA All bacilli are gram (-) except: MYCOBACTERIUM, CORYNEBACTERIUM, CLOSTRIDIUM, BACILLUS, ERYSIPELOTHRIX, LISTERIA, LACTOBACILLUS Higher forms of organisms like ACTINOMYCES , STREPTOMYCES, yeast and molds are gram (+)

Procedure of Gram Staining
Prepare a bacterial smear Overlay smear with Crystal violet for 30 sec ± 1 min Rinse with distilled water, tapping off excess Flood smear with iodine for 1 min Rinse with distilled water , tapping off excess Add acetone or ethyl alcohol drop by drop until no violet color appears in rinse . This requires less than 10 secs 7. Rinse with distilled water immediately 8. Flood smear with safranin for 30 secs 9. Rinse with distilled water and allow slide to drain 10. Blot dry and examine 1. 2. 3. 4. 5. 6.

ACID FAST STAINING ACID FAST ORGANISM ± these are organisms that are very hard to stain but once stained they are difficult to decolorize due to MYCOLIC ACID / HYDROXYMETHOXY ACID that envelopes the bacteria Rule : All bacteria are Non-Acid Fast except : Mycobacterium, Slightly Acid Fast is Nocardia

1. Steaming process 2. Increasing concentration of phenol blue and basic fuchsin 3. Prolonging contact of stain with the material 4. Addition of wetting agents ( tergitol ) to the stain solution

1. ZIEHL NEELSEN C ± carbol fuchsin A ± acid alcohol M ± methylene blue result : Acid Fast = Non-Acid Fast = RED BLUE

2. Kinyoun¶s/ Cold Method C ± carbol fuchsin A ± acid alcohol M ± malachite green * no steaming process Result : Acid Fast = RED Non-Acid Fast = GREEN

3. Pappenheim¶s ± differentiates M. smegmatis from M. tuberculosis 4. Baumgartens ± differentiates M. tuberculosis from M. leprae

Procedure of Acid Fast Staining ( Ziehl Neelsen )
1. 2. 3. 4. 5. 6. 7. Prepare a bacterial smear Flood smear with carbol fuchsin Gently steam over flame for 3-5 minutes( do not boil ) Wash off excess stain with water Decolorize for 15-20 secs with acid alcohol Counterstain with methylene blue for 1 minute Wash with water and allow it to dry and examine

Procedure of Negative Staining
1. 2. 3. 4. 5. Place a drop of nigrosin/india ink on a slide Add a loopful of culture on the drop Using the edge of another slide, spread the drop out Allow smear to dry but do not fix Examine bacteria appears colorless against a grayish black background

Result:

II. STUDY OF CULTURAL CHARACTERISTICS * Culture media CLASSIFICATION OF CULTURE MEDIA a) According to Physical State/Consistency 1. Liquid 2. semi-solid ² with 0.5 ² 1.5 % agar 3. solid ² with 1.5 ² 3.0% agar b) According to how media is dispensed 1. plated 2. tubed c) According to Use 1. general isolation media i.e. Nutrient Broth , BHI, TSB 2. Enrichment media i.e. Selenite broth, GN broth, tetrathionate broth

3. Selective media gentian violet methylene blue Na deoxycholate & other bile salts Vancomycin and penicillin potassium tellurite sodium azide alcohol chloral hydrate

inhibits gram positive organisms

inhibits gram negative organisms

PEA ( phenylethyl alcohol alcohol agar )- allows growth of gram positive cocci while inhibiting growth of gram negative

4. differential media ² i.e. BAP 5. Selective and differential media i.e. Mac conkey, EMB, XLD 6. Special media i.e. Fletcher·s ² Leptospira Bordet-Gengou ² B. pertussis

1. Culture the bacteria 2. Identify the cultured bacteria Methods of identification: a) morphological b) biochemical tests c) serological tests d) use of nwly discovered techniques like DNA hybridization 3. Test the susceptibility of bacteria to antimicrobial agents

INOCULATION TECHNIQUE IN THE CULTIVATION OF BACTERIA

1. 2. 3. 4. 5.

Liquid media Slanted media Butt media Butt/slant media Plated media 1. radial streak 2. overlap streaking 3. multiple streaking

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