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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