Bacteriology Gopal

8/11/2019 Bacteriology Gopal

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General Structure

Gram (-) Bacteria

Growth & Nutrition

Gram (+) Bacteria

Susceptibility Testing

Bacterial Diseases


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Gram positive bacteria have walls containing relatively largeamounts of peptidoglycan = a starch

Staphylococcus epidermidis , Streptococcus pyogenes , Clostridium tetani, Bacillus anthacis(ANTHRAX)


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Gram negative species have walls containing small amounts of peptidoglycan and a lipopolysaccharide = a fat/sugar combo

Escherichia coli , Salmonella typhi , Vibrio cholerae and Bordetella

Gram negative bacteria are harder to control with antibiotics


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Average size: 0.2-10.0 um in diameterBasic shapes

Cocci Bacilli

Spiral


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Elongated cocci: CoccobacilliExamples:

1. Listeria monocytogenes2. Haemophilus influenzae


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Unusual shapes Star-shaped Stella Square Haloarcula

Genetically, most bacteria are monomorphic (one shape)A few are pleomorphic based on environmental conditions


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Pairs: diplococci, diplobacilliPackets of four: tetrads

Packets of eight: octadsClusters: staphylococciChains: streptococci, streptobacilli


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Pallisade arrangement: Corynebacteriumdiphtheria


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MedicallyImportant Cocci

MedicallyImportant Bacilli

MedicallyImportant

Curved/Spiral Enterococcus spp. Neisseria spp.Streptococcus spp.

Enterobacter spp. Escherichia spp. Klebsiella spp. Proteus spp.Salmonella spp.Shigella spp.

Pseudomonasaeruginosa

Bacillus spp.Clostridium spp.

CURVED BACILLIVibrio choleraCampylobacter spp.(gull-wing)

SPIROCHETE

Treponema pallidum Borrelia spp. (Lymedisease & relapsing fever)


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Some bacteria may lose their characteristicshape because of adverse growth conditionsCell Wall Deficient Bacteria shapeless butrevert back to their original shape when placedunder favorable growth conditions.


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Pleomorphic

No cell wallHas the ability to exist in variety of shapesExample: Mycoplasma spp.


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It refers to the number of cells , not the cellsize.Growing microbes increases in number.


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Bacteria multiply by a process called binaryfission.The time required for the cell to divide or the

population to double is called generation(doubling) time .


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Stages of Binary Fission

Bacilli following division

Chromosome division, cell growth by lengthening

Chromosome divided, cell fully lengthened, growthof envelope,Chromosomes segregated

Cross wall completed

Daughter cells separate


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1. Lag phase : When the cells are adjusting to their newenvironment. During this phase, cellular metabolism isaccelerated, the cells are increasing in size, there is nocell division and therefore no increase in numbers .

2.Logarithmic (log)/Exponential phase: Underoptimum nutritional and physical conditions, the

physiologically robust cells reproduce at a uniform andrapid rate by binary fission


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3. Stationary phase: During this stage, the number ofcells undergoing division is equal to the number of cellsthat are dying. There is no further increase in cell numberand the population is maintained at its maximum level

for a period of time.

4. Decline or death phase : Because of the continuing

depletion of nutrients and buildup of metabolic wastes,the microorganisms die at a rapid and uniform rate.


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Nutritional Requirements

All organisms, whether they be bacteria, humans, or trees,need a constant supply of food in order to live. Water which is absolutely essential for cellular function. Carbon which is the major structural element in cellconstituents Energy required for cellular growth. Nitrogen which is also an important structural elements,

being a constituent of proteins & nucleic acids Traces of other elements required for life processes.


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All organisms, whether they be bacteria, humans, ortrees, need a constant supply of food in order to live.Fastidious organisms that has demanding nutritionalrequirements.


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Requirements For Growth1. Physical temperature, pH, osmotic pressure2. Chemical Water, sources of carbon & nitrogen,

minerals, oxygen & organic growth factors.


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TemperaturePsychrophiles cold loving microbes. Mesophiles moderate-temperature lovingmicrobes

Thermophiles heat loving microbes.


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Temperature RangeMinimum Growth Temperature : is the lowesttemperature at which species will growOptimum Growth Temperature : is the temperature

at which the species grows best.Maximum Growth Temperature : is the highesttemperature at which growth is possible


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Temperature PsychrotrophsGrow between 0 C and 20-30 CCause food spoilageAlso known as moderate psychrophiles or facultative

psychrophiles


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pHMost bacteria grow between pH 6.5 and 7.5.Molds and yeasts grow between pH 5 and 6 (greater

pH range compared to bacteria). Acidophilic Bacteria: remarkably tolerant of acidityBasophilic Bacteria: grows at pH near neutrality.


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Osmotic Pressure is the pressure that is exerted on a

cell membrane by solutions inside & outside the cell.What are the effects of the ff. solutions in a bacterial

cell?1. Hypertonic

2. Isotonic3. Hypotonic


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Hypertonic SolutionThe cell membrane & cytoplasm shrink away from the bacterial cell

wall Plasmolysis.Salts & Sugars are added to certain foods to preserve them.Bacteria in hypertonic environment will die as a result of

desiccation.


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Hypotonic SolutionIf a bacterial cell is placed in a hypotonic solution, thefluid pressure w/in the cell increases greatly.

If the pressure becomes so great & cell bursts,

cytoplasm escapes from the cell Plasmoptysis .


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Isotonic SolutionIn an isotonic environment, water neither leaves norenter the cell.


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Halophileshalo referring to salt & philic meaning to love Bacteria that loves salty environment.

Example: Vibrio cholera

HaloduricOrganisms that do not prefer to live in a salty

environment but are capable of surviving there.Example: Staphylococcus aureus


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Barometric Pressure

BarophilesOrganisms that thrive deep in ocean & oil wells,

where atmospheric pressure is very high.

Physical Requirements


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Water in the liquid state is essential for the existence of allliving organisms. Approximately 75% water is present in the cells of every livingorganism, including bacteria. This amount of water is required to maintain the cell in anactive state, and without liquid water, living organisms will not

be able to grow or reproduce. Substances such as sugar and salt makes the water unavailablefor bacteria. The amount of water available for microbial growth is referredto as Water Activity.


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Oxygen is essential for the growth of many bacteria, but for others it is lethal.

All bacteria have cell components that are sensitive

to oxygen and metabolic by-products of oxygen. Organisms that live in air have enzymes thatdetoxify these products.


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A. AerobicB. AnaerobicC.

FacultativeD. MicroaerophilicE. Aerotolerant anaerobe


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Obligate Aerobes absolutely require oxygen for their growth.

Example: Mycobacteria spp. Obligate Anaerobes are those that are unable to grow in the presence of free oxygen because O2 kills or inhibits them.Aerotolerant anaerobe does not require oxygen, grows better

in the absence of oxygen, but can survive in the presence ofoxygen.Facultative anaerobes are capable of surviving in either the

presence or absence of oxygen.

Examples: Enterobacteriaceae, most Streptococci &Staphylococci.Microaerophiles need a small quantity of oxygen, but largequantities inhibit their growth or even kill them.


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Chemical RequirementsCarbon Dioxide

An atmosphere of 5-10% CO2 is required by someorganisms, referred to as Capnophiles .Examples : Neisseria spp., Campylobacter spp.,

Haemophilus spp.


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Living State

To observe shape &arrangement of organismA drop of bacterialsuspension on slide,cover it with coverslip &focus.

Wet Mount


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Living State

To observe organismsmotility.A hanging drop slide w/concavity at the center isused.

Hanging Drop


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Fixed State Adhere organism onslide & apply stain.


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It is the process coloring the microorganisms with a dye thatemphasizes certain structures.
http://faculty.clintoncc.suny.edu/faculty/Michael.Gregory/files/Bio%20102/Bio%20102%20Laboratory/prokaryotes/P001087.JPG


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A thin film of asolution of microbeson a slide is a smear.

A smear is usuallyFixed to attach themicrobes to the slide

and to kill themicrobes.

Preparation ForStaining


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Stains consist of a positive and negative ion.

In a basic dye , the

chromophore is acation.

In an acidic dye , the

chromophore is ananion.


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Simple Stains

It is made up of an aqueous

solutionTo observe bacterial shape &arrangement.

A mordant may be used tohold the stain or coat thespecimen to enlarge it.


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Steps Color OfGram + Cells

Color OfGram Cells

Primary stain:

Crystal violet

Purple Purple

Mordant:

Iodine

Purple Purple

Decolorizing agent:

Alcohol-acetone

Purple Colorless

Counterstain:

Safranin

Purple Red


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Acid-fast StainAcid Fast stain binds only to those bacteria thathave a waxy material in their cell walls.It is used to identify Mycobacterium spp.

1. Mycobacterium tuberculosis2. Mycobacterium leprae *** Nocardia spp.


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Cells that retain a basic stain (bacteria thathave a waxy material in their cell walls) in the

presence of acid-alcohol are called acid-fastbacteria (stained red) .

Non acid-fast cells (stained blue) lose the basic stain when rinsed with acid-alcohol, and

are usually counterstained to see them.


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Special Stains Negative staining is the process in

which the background & not theorganism is stained.

Are used tocolor andisolate specific

parts of

microorganisms.


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Special Stains Heat is required to drive a stain

into endospores.

Are used to colorand isolatespecific parts ofmicroorganisms.


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Special Stains Flagella staining requires a

mordant to make the flagellawide enough to see.

Are used to colorand isolatespecific parts ofmicroorganisms.


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Culture Media: anything that possess nutritional &environmental requirements for bacterial growth.


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3 Types of CulturePure Culture made up of one specie of bacteriaMix Culture made up of organisms belonging todifferent species.Stock Culture pure culture of microorganisms as a

source of supply in the industry.


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Agar polysaccharide extracts of seaweed & commonly

used base medium.


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Esherichia coli on EosinMethylene Blue Agar (EMB).EMB is both a selective &differential media . It isselective for the growth of gram(-) bacilli


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Weigh Ingredients

Dissolve Ingredients

Adjust To Proper PH

Sterilization

Distribution In SterilePetri Dishes

In plated media : Sterile first before

distribution.

In tubed media : Distribute first beforesterilization.


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Techniques of Inoculation

Inoculation To introduce microorganisms into a culturemedium or host.

Liquid Culture Medium inoculate the organism & shake.Slant Tubed Medium streaking from the bottom with azigzag fashionButt Medium stabbingButt/Slant stab, then streakPlated Medium - streaking


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Methods of Streaking Plated Medium

Radial Streak Method place the inoculum one side ofthe plate, then bring on the other side concentric fashion.Overlap Streak Method keeps on overlapping, used in

sensitivity testing.Multiple Streak Medium divide the medium intoseveral division, then streak separatelyInterrupted Streak Method start streaking on one sideof the plate, stop, then turn for 180 degrees & streak againMultiple Interrupted Streak Method used to obtain

pure isolated colonies.


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Works well when the bacteria is present in high numbers.


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A pure culture contains only one species or strainA colony is a population of cells arising from a singlecell or spore or from a group of attached cellsA colony is often called a colony-forming unit (CFU)


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A colony contains millions of organisms. Size, shape, color,elevation & margin are observed to identify the bacteria.


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Size: pinpoint, small, moderate, or largePigmentation: color of colony.Form: The shape of the colony.

(1) Circular: unbroken peripheral edge.(2) Irregular: indented peripheral edge.

(3) Rhizoid: rootlike spreading growth.

Margin: The appearance of the outer edge of thecolony .(1) Entire: sharply defined, even.

(2) Lobate: marked indentations.(3) Undulate: wavy indentations(4) Serrate: toothlike appearance(5) Filamentous: threadlike, spreading edge


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Elevation: The degree to which the colony growth israised on the agar surface.(1) Flat: elevation not discernible.(2) Raised: slightly elevated.(3) Convex. Dome shaped elevation.

(4) Umbonate. Raised with elevated convex region


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Bacteriology Gopal

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