Postlab Discussions in Microbio1 Lab

8/3/2019 Postlab Discussions in Microbio1 Lab

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POSTLABDISCUSSIONS IN

MICROBIO1 LABCompiled by:

Fortune Lapira Torrecampo, RMT


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11/18/2011 copyright (your organization) 2003 2

MICROSCOPE AND OTHER

INSTRUMENTS


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RESOLUTION

Ability of the lens to distinguish fine detail of the

specimen

Determined by the wavelength of light from the

illuminator.

A wavelength is the distance between the peaks oftwo waves.

As a general rule, shorter wavelengths produce

higher resolutions of the image seen through the

microscope


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PHASE CONTRAST

MICROSCOPE

-Bends light that passes through the specimen so

that it contrasts with the surrounding medium

-Bending the light is called moving the light out of

phase

-Since the phase-contrast microscope compensates for

the refractive properties of the specimen, you dont

need to stain the specimen to enhance the contrast of

the specimen with the field of view

-This microscope is ideal for observing living

microorganisms that are prepared in wet mounted

slides so you can study a living microorganism.


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FLUORESCENCE MICROSCOPE

Fluorescent microscopy uses ultraviolet light to

illuminate specimens.

Some organism fluoresce naturally, that is, give off

light of a certain color when exposed to the light of

different color.

Organisms that dont fluoresce naturally can be stained

with fluorochrome dyes. When these organisms are

placed under a fluorescent microscope with an

ultraviolet light, they appear very bright in

front of a dark background.


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ELECTRON MICROSCOPE-Developed in the 1930s, the electron microscope uses

beams of electrons and magnetic lenses rather than

light waves and optical lenses to view a specimen.

-Very thin slices of the specimen are cut so that the

internal structures can be viewed.

-Microscopic photographs called micrographs are taken

of the specimen and viewed on a video screen.

-Specimens can be viewed up to 200,000 times

normal vision.

-living specimens cannot be viewed because the

specimen must be sliced.


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DARK FIELD MICROSCOPE

If the regularly used condenser is

replaced with what is known as a

darkfield condenser, illuminated

objects are seen against a darkbackground (or dark field), and

the microscope has been

converted into a darkfieldmicroscope


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MICROBIAL CONTROL

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Number of organisms initially

present

Killing effectiveness is gauged

by decimal reduction time

D value

Time required to kill 90%

of population under specific

Conditions

Effect of prior washing


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MICROBIAL CONTROLPhysical Methods

I. HEAT

- coagulates protein

A. Moist Heat

- water aids in the disruption of covalent bonds

1. Boiling

- 1000C 15 30 minutes

- kills vegetative forms but not spores and viruses

-Mechanism of Action: Denaturation

2. Fractional Sterilization2.a Tyndallization

- steam for 30 minutes in 3 consecutive days

2.b Inspissation

- 75 800C for 2 hrs. on 3 successive days



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3. Autoclaving

- steam under pressure

-1210C at 15 psi for 15 30 minutes


4. Pasteurization

- sterilization for milk

-620C for 30 minutes followed by rapid cooling (Flash

Pasteurization)


5. Inspissation

-75 to 800C for 2 hours fro 3 consecutive days (withincubation in between); used to sterilize culture media

with egg, serum, or sugars



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Pressurized steamAutoclave

Achieves sterilizationat

121C and 15psi in

15 minutes

Effective against

endospores

Prions destroyed at

132C

for 4.5 hours


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B. Dry Heat

- kills by oxidation

a. Hot air oven

- 160 1800C for 1 2 hrs

- time depends on the penetration of heat on

objects to be sterilized

- for metals and glasswares

-Mechanism of Action: Oxidation

b. Direct Flame

- usually for inoculating loops and needles

-Mechanism of Action: Burning contaminants to

ashes

C. Incineration

-used for swabs, dressings, wipes, etc.

-Mechanism of Action: Burning materials to ashes

II FILTRATION


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II. FILTRATION

- physical separation of microbes from liquid

Example:asbestos, Millipore, nitrocellulose

- Mechanism of Action: Physical removal of

bacteria from suspending liquid or gas

- Usually used for substances that could not

tolerate heat ( injectable drugs, vitamins, amino acids)

TYPES OF FILTERS FOR LIQUIDS:A. Membrane filters made of nitrocellulose or

cellulose acetate with uniform pore diameters of

0.45um or 0.22 um

B. Depth fileters made of porcelain, glass or fibrous

material (cotton, asbestos, and paper); microbesare retained by tortuous passageway

AIR FILTERS high efficiency particulate air (HEPA)

filter removes almost all microbes >0.3um in dm.

Used for specialized hospital rooms and in laminar

flow hoods


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Filtration

Liquid filtration

Membrane filters allow

liquids to flow through

Filtration of airHigh efficiency

particulate air (HEPA)

filter removes nearly allmicrobes from air

Filter has 0.3m pores

to trap organisms


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III. OSMOTIC PRESSURE

-Mechanism of Action: by plasmolysis

Example: immersion of meat in salt solution

Fruits and vegetables in sugar solution

-results in loss of water from microbial cells

-Used in food preservation

IV. SONIC VIBRATION, TRITURATION,

AGITATION

- mechanical methods that disintegrate bacteria

Sonic vibration sound waves

Trituration grinding

Agitation shaking


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V. COLD

- Mechanism of Action: Decreased chemial

reactions and possible changes in proteins

- used in the preservation of food, drug,

and culture

Refrigeration bacteriostatic effect

Deep freezing- between -500

C and -950

CLyophilization water removed by high

vacuum at low temperature; for long tern

preservation

VI. DESSICATION- Mechanism of Action: Disruption of

metabolism

- Used in food preservation, bacteriostatic,

- Removes water from microbes


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VII Radiation

Electromagnetic radiation

Energy released as waves

(microwaves, radio, gamma, X rays, ultraviolet light etc)

Shorter wavelength, higher frequency = more energy

Radiation can be ionizing or non-ionizing


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Ionizing radiationGamma radiation

X-raysElectron accelerators

Causes direct damage to DNA and potentially to

cell membrane

Causes indirect damage by producing reactive

molecules such as superoxide and hydroxyl radicals

Used to sterilize heat sensitive materials Medical equipment, surgical supplies, medications

Some endospores can be resistant


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Ultraviolet radiation

Non-ionizing (NI) radiation

Damages DNA (causes thymine dimers)

Used to destroy microbes in air, drinking water and

on surfaces

Limitation

Poor penetrating power (thin films or coverings can limit itseffectiveness)


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Chemicals

Chemicals can be used

to disinfect and sterilize

Called germicidal

chemicals

React with vital cell

structures and components

Proteins

DNA

Cell membrane


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Potency of chemicalsFormulations generally

contain more than one

antimicrobial agent

Regulated by FDA

Antiseptics

EPADisinfectants

Grouped according to

potency

Sterilants Destroy all microorganisms

High-level disinfectants Destroy viruses and vegetative

cells but not endospores

Intermediate-level disinfectants

Kill vegetative cells fungi, mostviruses but not endospores

Low-level disinfectants Kill fungi, vegetative bacteria and

enveloped viruses

No effect on mycobacteria, nakedviruses or endospores

II Ch i l M th d


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II. Chemical MethodsANTISEPTIC

a chemical substance that prevents growth of bacteria by

either inhibiting or destroying microbes

used on the surface of skin or mucous membranes

DISINFECTANT

kills many but not all microbes aims to kill disease-

causing microbes but not spore formers used on inanimateobjects

BACTERIOSTATIC

agents that inhibit the growth of bacteria

BACTERICIDE

agents that kill bacteria

SANITIZERa ents that reduce bacterial numbers to safe levels


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VARIABLES OF DISINFECTION

1.Concentration

2. Time3. Temperature

4. pH

N 1 where: N = no. of surviving bacteria

CT C = concentration

T = time

Increased concentration

Increased Time Decreased Survivors

CHEMICAL AGENTS


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CHEMICAL AGENTS

A. Disruption of Cell Membrane

Alcohols

- denatures protein

- disorganizes the lipid structure in the membranesExample: 70% isopropyl alcohol

- requires water for maximal activity

Detergents/ soaps- surfactants interact with the lipid in the cell membrane

and with the surrounding water

- increases the surface tension

Example: Quaternary ammonium (Quats or Zephiran)

Phenols

- original disinfectant of Lister

- denatures protein

Example: carbolic acid/ cresol (Lysol)


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B. Inhibition of Protein Synthesis

1. Chloramphenicol

- from Streptomyces venezuelae

- for Gram + and Gram - bacteriostatic

Disadvantage:suppresses the bone marrow (anemia)

2. Erythromycin

- freezes the ribosome3. Tetracycline

- broad spectrum

4. Streptomycin

- from Streptomyces griseus

- for MTB and N. gonorrhoeae

5. Gentamycin, Lincomycin, Clindamycin andAminoglycosides


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C. Injury to Cell Membrane

1. Amphotericin B

2. Polymyxin B- from Bacillus polymyxa

- for Gram

Example: Brucella abortus, Klebsiella pneumoniae

3. Tyrocidin

4. Garamicidin S

5. Imidazole and Triazole


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D. Inhibition of Nucleic Acid Synthesis

1. rifampin

2. Quinolones

3. Sulfonamides

4. Trimethoprim


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DIRECT EXAMINATION OFUNSTAINED SPECIMENS



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SALINE MOUNT

A G G DROP


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HANGING DROP

PREPARATION


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MOTILITY

TRUE MOTILITY

BROWNIAN MOVEMENT



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CYTOLOGY



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Bacterial Morphology

SHAPE Cocci in Latin it means berry,

spherical in shape

Bacilli it means little stick or rod

in Latin, rod shaped Spiral rod shaped with convolutions

Vibrio curved rods that resemble acomma in serpentine S shape

Spirilla spirals or corkscrew in shape

Spirochetes spiral with an ability towriggle or flex

Pleomorphic with no definite shape


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ARRANGEMENT1. Singles

2. Pairs diplococci, diplobacilli

- cell division occurs in a single plane

3. Chains Streptococci, Streptobacilli

- divides on 2 planes end to end4. Clusters staphylococci (grape-like)

- divides in 4 or more planes

5. 4s tetrads

- divides in 2 planes

6. 8s sarcina- divides in 3 planes

7. Pallisade side-by-side division

- picket fence in appearance


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PROKARYOTES AND EUKARYOTES

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The Bacterial Cell and itsStructures

I Cell Wall


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I. Cell Wall- maintains cell shape

- determines if the bacteria is Gram + or Gram

- protects against osmotic pressure change

a. Peptidoglycan / Murein or Mucopeptide- thicker in Gram +- susceptible to tears, saliva and mucus due to thepresence of the lysozyme

Functions:

1. maintains integrity of the cell wall

2. target of some antibioticsEx. Penicillin

*Protoplast

- seen in bacteria whose peptidoglycan layer

is removed but are still viable, this makes themresistant to penicillin


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b. Teichoic Acid

- protein attached to the peptidoglycan layer

- found in Gram + only

Functions:Antigenic

c. Outer Membrane- found in Gram negative only

- composed of lipopolysaccharide (LPS),lipoprotein and phospholipids

***Periplasmic space

- space between the outer membrane and theplasma membrane


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Other Properties:

1. Endotoxin (LPS)

2. Antigenic

- capable of producing Antibodies

Ex. Streptococcus pyogenes (ASTO)

Salmonella typhi (Typhidot)

3. Contains Proteins

- regulates the passage of materialsfrom inside to outside


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II. Cytoplasmic Membrane / PlasmaMembrane- similar to eukaryotes (phospholipids bilayer)

- fluid mosaic model

Functions:

1. Active Transport- requires energy

- movement is against concentration gradient

2. Energy generation

- oxidative, phosphorylation

3. Synthesis for the precursors of the Cell Wall

4. Secretion of enzymes and toxins

**Mesosomes

- invaginations of the cell membrane

C l i b


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II. Cytoplasmic Membrane /Plasma Membrane (continued)

Functions: shown in cell division

binding site of DNA

III C t l


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III. Cytoplasm- contains the Cytosol or the Amorphous Matrix to which nearly all

other functions not conducted by the cell membrane occur

- it is where interior structures are suspended

CONTENTS:

1. Ribosomes- for protein synthesis

- it is significant because it is the target of some antibiotics

2. Granules- storage sites of nutrients

- also important for staining purposes

Ex. Corynebacterium - Babes-EarnstMycobacterium - Muchs

Yersinia - Bipolar bodies

3. Nucleoid- single circular molecule of DNA

- the essence of a Prokaryote

III C t l ( ti d)


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III. Cytoplasm (continued)4. Plasmids

- extrachromosomal circular molecule of DNA

- it can replicate by themselves independent from the chromosomes

- it can be integrated into the DNA of another nucleoid

2 Types of Plasmids1. Transmissible

- transferred from one bacteria to another thru CONJUGATION by the pili2. Non-transmissible

Functions:

1. Antibiotic Resistance

- this is the reason why resistance of bacteria to antibiotics are carriedfrom one bacteria to another

2. Resistance to heavy metals

3. Resistance to UV light

III C t l ( ti d)


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III. Cytoplasm (continued)

5. Transposons- also known as Jumping Genes

- these are short straight

extrachromosomal molecule of DNA

Functions:

1. Drug resistance2. Mutations

S i li d St t


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Specialized Structures

1. Capsule

- an excretory product that is polysaccharide in natureImportance:

a. Virulence- limits phagocytosis

- encapsulated bacteria are pathogenic

b. Identification

- in the Neufeld-Quellung Reaction for Streptococcuspneumoniae

c. Production of Vaccines- due to its high antigenic properties

d. Adherence or Attachment

S i li d St t


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Specialized Structures(continued)

2. Flagella

- contains the proteinflagellin

- requires energyFunction: Locomotion

-

observed thru thehanging drop slide orin biochemical tests

Importance:

a. Chemotaxis

- attraction to chemicalstimuli

b. Identification

Arrangementa. Atrichous

- no flagellum

b. Monotrichous- 1 flagellum atone pole

c. Lophotrichous- tuft of flagellaat one pole

d. Amphitrichous- tuft of flagellaat both poles

e. Peritrichous- flagellated allover



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3. Fimbriae or Pili

- straight and short appendages

- contains the protein pilin

- seen in Gram bacteria

Importance:

a. Attachmentb. Sex Pilus

- for conjugation



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4. Glycocalyx or Slime Layer- composed of loose

polysaccharides

Function:adherence orattachment

Ex. Streptococcus mutans



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Spec al ed St uctu es(continued)

5. Spores

- formed in response to adverse conditions in theenvironmentFunction: Protection

Processes of Sporulation1. Axial Filament formation

2. Forespore Septum formation- infolding of the cell membrane toproduce a double membrane

3. Engulfment of Forespore- results to the synthesis of 2 special

layers forming the cell envelope

4. Cortex Synthesis5. Coat Deposition6. Maturation7. Lysis of Mother Cell


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SPECIMENCOLLECTION

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Microbiological examination


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Specimens for microbiological examination must beappropriate eg, sputum rather than saliva.

In general, specimens should be collected into, andtransported in, a sterile container.

Aspirated pus may be transported in a syringe, which mustbe capped immediately the needle has been removed and

disposed of safely.

Specimens should be delivered promptly to the laboratory.Although many specimens will tolerate a delay of several

hours if refrigerated, cerebrospinal fluid must be transported

to the laboratory immediately, without refrigeration.

Similarly, for the detection of Neisseria gonorrhoeae and

other fragile organisms, special arrangements may beneeded: eg, express delivery, inoculation of plates at the

time and place of collection, provision of special transportcontainers.

Special requirements, for individual tests, are noted in the

Test listing

Microbiological examination

Tube Guide


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Tube content Determination Instructions Shape

Heparin

Produce blue background inblood smeer

Plasma testing

some general chemistry(Glucose, urea,Cr)

Invert slowly several times

to ensure mixing

EDTAInhibit ALK, CK

Unsuitable for Ca&coagulation

Routine hematology ,

blood cont, Retc. CT.

Sickle test, Glyco HB,

HBelectro, ACTH,AS

Invert slowly several times

to ensure mixing

Plain, No additive Hormones, Generalchemistry

Blood group, RH, Cross

match,, Serology &Allergy,

Sodium Citrate1:9Calating Ca,Inhibit

aminotransferase. ALK &

stimulate ACP

Fibrinogen, PT, PTT, TT,

ATIII, coagulation Screen

Ensure tube fills correctly

to volume on label

Sodium Citrate1:4 ESR Ensure the presence ofanticoagulant Invert slowly
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anticoagulant, Invert slowly

several times to ensure mixing

Plane Urine &Stool

analysis,

Plane Urine &

Stool

culture

Media for

maintenance ofbacteria

Blood 1- clean the area from which the

blood is collected by iodine2- withdraw for 8-10 ml of blood

3- insert the blood immediately in

the vial and bring it to the lab

quickly (2-3 ml for children and

5-7 ml for adult)

Type of urine specimens


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1) Random specimens (drug abuse)

2) First-morning (microscopic examination, b-

HCG, 8-hours)

3) 24-hours specimen

Some analyses produce in different time though 24 hours of

collection morning or noon like Creatinine, protein, Ca, phosphorsand electrolyte (the sample must be refrigerated)

4) clean-catch specimen (MSU) for bacterial culture

5) catheter specimen6) suprapubic specimen especially for infant

7) urine collected from children

collection bags with hypoallergenic skin adhesive


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If the sample left at RT

if the organism urase producer, ammonia release willincrease Ph resulting in destruction of cells and cast

the bacteria will break down any glucose

RBC, WBC, Cast will lyze

Protein conc will alter Bilirubin and Urobilinogen oxidized-not detected

Uric acid and urate deposited to for oxalate or phosphatecrystal

normal bacteria will multiply producing contaminated sample

Microbiological sample


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specimen container instruction

sputum Clean,

wide nick

Early morning, cough deeply for

sputum, for children gastric wash,delay of specimen TB, Pn, HI

Throat swab Sterile

swab

Not contaminated with saliva, no

antibiotic by 8-hours

stool Wide nick Must be fresh sample within 2

hours

Rectal swab For

cholera

alkaline

peptone

waterBlood culture Bldculture

bottle

Take the sample when tempt high, use

iodine for sterilization, mix it and must

reach the lab early, never refrigerated

semen Clean and

sterile

3-7 days of sexual abstinence, no

condom, time of collection and

deliver to the lab within 2 hour


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Criteria for rejection of specimens

oMissing or inadequate identiification

oInsufficient volume

oSpecimen collected in wrong collection tube

oContamination

oInappropriate transport and storage

oUnknown time delay

Oropharyngeal (Throat) Swab


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Oropharyngeal (Throat) Swab

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Transporting Specimens


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Transporting Specimens

from Field to Lab

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Gram staining


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Gram staining

- a principal stain

- most clinically significant bacteria are detected

except:

a. intracellular bacteria

b. bacteria that lacks cell wall

c. bacteria with insufficient dimensions to be

resolved by light (i.e. spirochetes)- provides preliminary diagnosis for initial treatment


G t i i


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Gram stainingPrinciple

- Gram (+) bacteria have thicker peptidoglycan layer

(40) with numerous teichoic acid cross-linkages thanthat of G (-) (1or 2)

- teichoic acid prevents decolorization

- G(+) bacteria may loose CW integrity by:

- antibiotic treatment

- old cells

- use of autolytic enzymes

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Gram staining


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Gram stainingRule:

1. All cocci are Gram (+) except:

Neisseria

Branhamella

Veilonella

2. All bacilli are Gram(-) except:Bacillus

Clostridium

Corynebacterium

Erysipelothrix

LactobacillusListeria

Mycobacterium



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Gram staining

Reagents

Crystal Violet - initial stain

Grams Iodine - mordant

95% Alcohol - decolorizer

Safranin - counter satin

Gram (+) - stains BLUE / VIOLET

Gram (-) - stains RED / PINK



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Acid Fast Staining

other commonly used stain for light

microscopy

- for Mycobacterium species

Principle

- designed for bacteria whose cell wall

contains long chain fatty acids (mycolic

acid)

- mycolic acid render the cells resistant to

decolorization

- Acid fast organisms may be Gram (+)



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Acid Fast StainingReagents

1. Carbol Fuchsin- initial stain2. Acid Alcohol - decolorizer

HCl + ethyl alcohol

3. Mehtylene Blue - counter stain or Malachite Green

a. Hot Method Ziehl - Neelsen

- uses steam as a mordant

b. Cold Method Kinyoun Modification

- uses phenol or tergitol as mordant

Acid Fast Bacilli- stains RED or PINK

Non-Acid Fast Bacilli- stains BLUE or GREEN

Postlab Discussions in Microbio1 Lab

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