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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.
Autotrophic Heterotrophic
Halophilic
Capnophilic
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 growth rate 4. Death Phase/Phase of Decline 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
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, Anthonys ) 3.2 Spore stains ( Dorners , Schaeffer and Fulton, Wirtz conklin ) 3.3 flagellar stain ( Grays, Fisher & Conn, Leifson ) 3.4 metachromatic granules ( Alberts, 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 (+)
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. Kinyouns/ Cold Method C carbol fuchsin A acid alcohol M malachite green * no steaming process Result : Acid Fast = RED Non-Acid Fast = GREEN
3. Pappenheims differentiates M. smegmatis from M. tuberculosis 4. Baumgartens differentiates M. tuberculosis from M. leprae
Result:
II. STUDY OF CULTURAL CHARACTERISTICS * Culture media CLASSIFICATION OF CULTURE MEDIA a) According to Physical State/Consistency 1. Liquid 2. 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. 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
PEA ( phenylethyl alcohol alcohol agar )- allows growth of gram positive cocci while inhibiting growth of gram negative
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
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
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