Chapter 8 Introduction to Laboratory Technique.pptx

8/11/2019 Chapter 8 Introduction to Laboratory Technique.pptx

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DNS 0303

Microbiology & Parasitology

Chapter 8

Introduction to Laboratory Technique


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What is microscope?

Different types of microscope

Parts and uses of microscopes

Procedures of using microscope

Handling of microscope

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Contents


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What is inoculation?

Different types of Culture medium / Growth

medium

Aseptic techniques

Inoculation of culture media

Principle of stainingWhat is simple staining, Gram staining and

acid fast staining?


Contents (Cont.)


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Upon completion of this chapter, the

students will be able to:

(a) Describe the differences between light

and electron microscope.

(b) Discuss the parts and uses of

microscope.

(c) Briefly explain how to observe

specimen by using a light microscope.

(d) Describe how to handle the

microscope. 11/10/2014DNS 0303 Microbiology & Parasitology4

Learning Outcomes


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(e) Discuss the inoculation of culturemedia.

(f) Define, describe and give examples of

the different kinds of media.(g) Explain the uses of various types ofmedia.

(h) Explain the importance of aseptictechniques.

(i) Describe the principle of staining anddifferences between simple staining,

Gram staining and acid fast staining. 11/10/2014DNS 0303 Microbiology & Parasitology5

Learning Outcomes (Cont.)


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Micro = small Scope= to view

A scientific instrument with one or more lensesthat allow you to observe small specimens which

is not visible to the naked eye.

It magnifies the image of the object to bevisualized through it.

Normally the laboratory microscopes provide amagnification of 40X (scanner), 100X (lowpower), 400X (high power) & 1000X (oilimmersion)


What is microscope?


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What is microscope?


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Different Types of Microscope

Types of microscope

Light / Opticalmicroscope

Bright-field microscopewellsuited for viewing stainedspecimens, eg. Stained blood

smears Phase-contrast microscope

Epi-fluorescence microscope

Electron microscope Transmission electron

microscope (TEM)

Scanning electronmicroscope (SEM)


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Optical microscope

(a) function through the optical theory oflenses in order to magnify the image

generated by the passage of a wavethroughthe sample

(b) most simplest & most widely usedtype of microscope

(c) typical magnificationof a lightmicroscope is up to 1500x


Different Types of Microscope (Cont.)


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Electron microscope

(a) uses electrons to illuminate a

specimen & create an enlarged image

(b) have much greater resolving power

than light microscopes

(c) can obtain much higher

magnifications (~ 2 millions X)




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Variants of Electron microscope

(a) Scanning Electron Microscope (SEM)

- looks at the surface of bulk objects by

scanning the surface with a fine electronbeam & measuring reflection

(b) Transmission Electron Microscope(TEM)

- passes electrons completely throughthe sample, analogous to basic optical

microscopy




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Different Types of Microscope

(Cont.)


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Parts of Microscope (Cont.)


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Ocular / eyepiece- monocularor binocular

- the ocular / eyepiece are located at the topof the microscope, are attached to a barrel ortube connected to the microscope arm- each ocular, through which the object isviewed, contains a magnifying lens.

- Usual magnification = 10X




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Objective lens- underside of the microscope armcontains a revolving nosepiece towhich the objectives are attached

- at least 3 objectives(a) lowpower objectives:

magnifies 10X

(b) highpower objectives:

magnifies 40X(c) oil immersion objective:

magnifies 100X




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To determine the degree of

magnification, the magnification listed on

the ocular (10X) usually is multipliedby

the magnificationlisted on the objectivebeing used.

Eg. Object viewed on 10X ocular & high

power 40X = would be magnified 400X11/10/2014DNS 0303 Microbiology & Parasitology16


Total magnification = magnification of objective x magnification of eyepiece


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Light source, condensor & diaphragm

- The microscope arm connects theobjectives& eyepieceto the microscope

base, which supports the microscope.- The base also contains the light, whichilluminatethe object reviewed. Locatedabove is the moveable condenser & iris

diaphragm.- Condenser: focuses or directstheavailable light into the objectiveas it israised or lowered & enhances specimen

contrast. 11/10/2014DNS 0303 Microbiology & Parasitology17



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Coarse adjustment

- focus with low-power objective only

Fine adjustment- give a sharper image after the object isbrought into view with the coarse adjustment.

The higher the magnification of theobjective, the shorterthe working distancewill be.




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Stage

- is supported by the arm & is located

between the nosepiece & the light

source

- serves as the supportfor the object

being viewed & has stage clip to keep

slides stationary




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Light microscope

(a) Low power:

- examine small living animal & plant

cells

(b) High power:

- examine bacteria- view drug particles / shape of the

crystals


Uses of Microscope


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Electron microscope

(a)image, characterize & manipulate

material structures at exceedingly small

scales including features of atomicproportions.


Uses of Microscope (Cont.)


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1. Turn the revolving nosepiece to engage the10X objective. Make sure that the revolving

nosepiece stopswith an audible click.

2. Lowerthe stageusing the coarse adjustment& gently place a prepared slide on the stage in

the specimen holder clips. The specimen holder

clips are spring loaded & if forcibly

released may break the slide.


Procedures of using Microscope


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3. Turnthe mechanical stage controls to adjustthe positionof the slides. Do not move the

stage manually without adjustment knobs.


Procedures of using Microscope (Cont.)


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4. Switchthe main switch to ONand adjustthebrightnesswith the light intensity knob.

5. Lookthrough the eyepiece, turnthe coarse

adjustment knob to bring the specimen intofocus. When you have optimized the focus

with the coarse adjustment controls,

use the fine adjustment knob

to improvethe focus.




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6. Start with the most light & gradually lessen ituntil the specimen imagehas clear, sharp

contrast. Then, adjustthe diaphragmto get

the best lighting.

7. Scanthe slide(right to left & top to bottom) at

low power to get an overview of the specimen.

Then centre the part of the specimen you wantto view at higher power.




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8. Engagethe objectiveto be used forobservation by turningthe revolving

nosepiece. Refocusby rotating the coarse

adjustment knob to reach the pre-focusing

position. This is limited by the engaged pre-

focusing lever. Make fine adjustments with the

fine adjustment knob. Always focus up,

moving the objective away from the slide.




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9. Rotatethe nosepieceto the 10X objective for100X magnification. Refocus & view your

specimen carefully. Adjustthe lightingagain

until the image is most clear (you will need

more light for higher power). Repeatwith the

40X objective for 400X magnification, which

will enable you to see all of the specimen detail

that is necessary for high school biology labwork.




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Handling of microscope

First clear your desk to receive the microscope, then graspits arm firmly, lift & support under the base with otherhand, set on a cleared desk.

Remove & store its dust over in cabinet under desk.

Unwrap power cord, loop once around gas outlet at rear ofdesk, plug into electrical outlet in front of desk.

To carry a microscope

Use ONLY lens paper.

Polishthe objectives & oculars.

Clean the lenses

Always begin slide set-up with the stage lowered & the lowestpower objective (4x) in place.


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Handling of microscope (Cont.)

Focus initially only by LOWERINGthe stage to thefocal point using the coarse focus.

Make only minor changesin focus whennecessary with the fine focus knob.

If you totally lose focus, returnto a lower

power objective to find the focal point. Do not use the 100X objective unless you have

received specific instructions on its use.

Use only the fine focus with higher powerobjectives.


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Handling of microscope (Cont.)

Clean the lens

with lens paper


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The act of introducing microorganism /suspensionof microorganisms (eg.Bacteria) into a culture medium

Theintroductionof microorganisms into a

growth medium, to cause the growth&multiplication of the microorganisms


What is inoculation?


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Inoculum:

- microbeswhich are introducedinto a

culture medium to initiate growth

- also known as inoculant

Culture:

- Microbesthat grow& multiply on a

culture medium


Definition


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= A nutrient material prepared for thegrowthof microorganisms in a

laboratory

Depending the special needs of particular

bacteria, a large variety& types of

culture mediahave been developed withdifferent purposes& uses.


Culture medium


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Culture media are employed in theisolation&maintenanceof pure

culturesof bacteria & are also used for

identification of bacteria according totheir biochemical& physiological

properties


Culture medium (Cont.)


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Type ofmedia

Agar Broth


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Solidmedium

A complex polysaccharidederived from a

marine alga

Usually contained in test tubes or Petri

d ishes

Liquid media are often mixed with agar &

poured into petri dishes to solidify.


Agar


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Types of agar media

Types of agar media

Chemically defined media

Complex media

Selective media

Reducing media

Differential media

Enriched media


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Types of agar media (Cont.)


Exact chemical composition is known

Chemoheterotroph& autotrophic (directlyuse sources of energy - light to produceorganic substrates from inorganic CO2)

Contain organic growth factors that serve

as a source of carbonand energy


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Eg. Glucose, Ammonium phosphate,

sodium chloride, magnesiumsulphate, potassium phosphate andwater is included in the medium for

growing Escher ich ia co l i Other: Neisseria,Lactobaci l lus


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Complex media

Heterotrophicbacteria & fungi

Made up of nutrient including extracts fromyeasts, meat or plants / digests of proteins

Energy, carbon, nitrogen & sulphurrequirements of the growing

microorganisms are primarily provided byprotein


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Complex - constituents

Peptone (partially digested protein)

Beef extract

Sodium chloride

Agar

Water


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Complex media

If a complex medium is in liquid form, it iscalled nutrient broth.

When agar is added, it is called nutrient agar.

Also known as undefined medium

Why?amino acid source contains a variety of

compounds with the exact composition beingunknown


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Complex media Nutrient agar


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Selective media

Is used to suppress the growth of unwantedbacteria& encouragethe growth of the

desired microbes Eg. Bismuth sulphite agar

- isolate the typhoid bacterium, gram-negativeSalmonella typhi from faeces

- inhibits gram-positive bacteria and most gram-negative intestinal bacteria


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Selective media

Eg. Sabouraudsdextrose agar (SDA)

- pH 5.6

- isolate fungi that outgrow most

bacteria at this pH


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Selective media

Sabouraudsdextrose agar


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Reducing media

Anaerobic bacteria which might be killed

by exposureto oxygen = Anaerobic Growth Media

Ingredients: sodium thioglycolate, whichchemically combine with dissolved oxygen

& deplete the oxygen in the culturemedium


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Differential media

Distinguish colonies of the desired organism

from other colonies growing on the same plate Eg. Blood Agar (contains red blood cells)

- identify bacterial species that destroy redblood cells

- Streptococcus pyogenes : causes sore throat,show a clear ring around their colonies - lysed


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Differential

media

Staphyloco ccus aureus colonies on BA - The bacteria have lysed the red blood cells,

causing the clear areas around the colonies


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Differentialmedia

Staphylococcus epidermidis colonies on BA


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Differential media

Eg. Eosin Methylene Blue (EMB) media

- differential for lactoseand sucrosefermentation

- inhibits the growth of Gram-positive bacteriaand provides a colour indicator distinguishingbetween those organisms that ferment lactose

versus those that do not. Organisms whichferment lactose display "nucleated colonies" --colonies with dark centres


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Differential

media

Escherichia coli on EMBmedium


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Selective & Differential media

Sometimes, selective & differential characteristics arecombined in a single medium

Eg. MacConkey agar (MAC) - selective and differential media used to differentiate

between Gram negative bacteria while inhibiting the growthof Gram positivebacteria. The addition of bile salts andcrystal violet to the agar inhibits the growth of most Gram

positive bacteria, making MacConkey agar selective.Lactose and neutral red are added to differentiate thelactose fermenters, which form pink colonies, from lactosenonfermenters that form clear colonies.


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Mac Conkey (MAC)agar


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Mac Conkey agar

E. coli and Proteus on

MAC


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Enriched

Bacteria present in small numbers can be missed,especially if other bacteria are present in much larger

numbers Liquid

Provides nutrients& environment conditions thatfavour thegrowthof a particular microbe but notothers

Also a selective medium, but it is designed toincrease very small numbers of the desired type oforganism to detectable levels


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Enriched

Isolate from a soil sample a microbe that

can grow on phenol & is present in muchsmaller numbers than other species

If the soil sample is placed in a liquidenrichment medium(phenol is the only

source of carbon & energy), microbesunable to metabolize phenol will not grow


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Type Purpose

Chemically defined Growth of chemoautotrophs and

photoautotrophs; microbiological assays

Complex Growth of most chemoheterotropic

organisms

Reducing Growth of obligate anaerobes

Selective Suppression of unwanted microbes;

encouraging desired microbes

Differential Differentiation of colonies of desired

microbes from others

Enrichment Similar to selective media but designed to

increase numbers of desired microbes to

detectable levels


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Broth

Types of broth

Peptone water

Nutrient broth

Tryptic soy broth

Selenite-F broth


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Types of broth media

Peptone water

Non-selective enrichment medium

Coagulase test

Can be used for fermentation

studies with variouscarbohydrates

f (C )


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Types of broth media (Cont.)

Nutrient broth

Complex medium in liquid form Contains nutrients, vitamins,

minerals and other growth factors

Suspension of microorganisms

T f b h di (C )


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Tryptic soy broth

Basic medium used for culturing many

kinds of microorganisms Tryptic soy broth is used mostly togenerate a large supply of bacteria forcertain biochemical tests.

It can also be used in the determination ofbacterial numbers.

T f b th di (C t )


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Tryptic soy broth



T f b th di (C t )


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Selenite-F broth (SF)

For the isolation and cultivation ofSalmonellaspecies from faecesand other specimens.

A ti T h i


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Aseptic Techniques

A ti T h i (C t )


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Before inoculation with the desiredmicroorganisms, microbiological media &all materials coming into contact with itmust be sterile.

During any subsequent handling of thebacterial cultures, unwanted /

contaminant organisms must beexcluded employing aseptic techniques.


Aseptic Techniques (Cont.)

A ti T h i (C t )


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When handling specimens / cultures,aseptic technique is important to avoid

their contamination& to protect the

worker from infection.

In-house training to demonstrate the skills

of aseptic technique should be given tostaff who will process specimens /

cultures.



A ti T h i (C t )


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Sterilizationimplies the completedestruction of all microorganisms

including spores.

This is accomplished by the use of(a) heat

(b) chemicals

(c) radiation

(d) filtration



I l ti f C lt M di


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The streak plate method works wellwhen the organism to be isolated ispresent in large numbers relative to thetotal population.

However, when the microbe to be isolatedis present only in very small numbers, its

numbers must be greatly increasedbyselective enrichment before it can beisolated with the streak plate method.


Inoculation of Culture Media

I l ti f C lt M di


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The object of any streaking pattern is thecontinuous dilution of the inoculum to give

many well isolated colonies.

For multi-phase streaking it is crucial to

flamethe loopbefore starting the next

phase. Note the slight overlap into theprevious phase to pick up a small

inoculum.



I l ti f C lt M di


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1. Flame the loop and wire &streak a loopful of broth as at

A in the diagram.

2. Reflame the loop & cool it.

3. Streak as at B to spreadthe original inoculum over

more of the agar.

4. Reflame the loop & cool it.

I l ti f C lt M di


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5. Streak as at C.

6. Reflame the loopand cool it.

7. Streak as at D.

8. Label the plate andincubate it inverted.

I l ti f C lt M di


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Inoc lation of C lt re Media


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Most bacteria grow well at thetemperatures favoured by humans.

Mesophiles, with an optimum growth

temperature of 25 to 40C, are the mostcommon type of microbe.

The optimumtemperature for manypathogenic bacteria is about 37C, and

incubators for clinical cultures are usuallyset at about this temperature.





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After inoculation, the specimen should beretained for at least 48 hours after the

laboratory has issued the final report.

Most positive cultures plates can be

discarded within 2448 hours of issuing

a final authorized report.



Smear


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In preparation of staining, a small sampleof microorganisms is placed on aslide &

permitted to air dry.

The smear is heat fixed by quicklypassing it over a flame.

Heat fixing killsthe organisms,makes

them adhereto the slide& permitsthemto accept the stain.


Smear


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Principle of Staining


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Bacteria have nearly the same refractiveindex as water.

Therefore, when they are observed under amicroscope they are transparent or nearly

invisible to the naked eye.

Different types of staining methods are usedto make the cells & their internal structures

more visibleunder the light microscope. The cells are then visible against a light

background


Principle of Staining

Stains


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Stains

Basic stains

Cationic(positivelycharged), react with

material that isnegatively charged.Bacterial cell walls

have a slightnegative chargeattract & bind with

basic dyes

Eg.Crystalviolet,

safranin,basic

fuchsin,methylene blue

Acidic stains

Negativelycharged

chromophores& are repelledby the leavethe microbetransparent

Eg.Nigrosin &

congo red


Stains

Simple Staining


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Simple stains use one dye that stains thecell wall.


Simple Staining

Differential Staining


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Differential Staining

Differential

Stain

Gram stain Acid fast stain

Differential stains use two or more stains& categorize cells into groups

Gram Staining


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4 different reagents are used & the resultsare based on differences in the bacterial cellwall.

Gram positive bacteria have a relatively

thick cell wall composed of a specialcarbohydrate called peptidoglycan

Gram negative bacteria have a much

thinner cell wall composed of the samecarbohydrate, peptidoglycan, but with certainchemical differences, eg. the presence oflipopolysaccharides (LPS)


Gram Staining

Gram Staining (Cont )


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Gram Staining (Cont.)



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Procedures1. A heat-fixed smear is covered with a basic

purple dye (crystal violet). Because thepurple stain imparts its colour to all cells, it is

referred to as a primary stain.

2. After 1 minute, the purple dye is washed off,& the smear is covered with iodine, a

mordant (1 minute).When the iodine is washed off, both Gram

positive & Gram negative bacteria appear darkviolet/purple.





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3. Next, the slide is washed (3 10 sec) withalcohol / alcohol-acetone solution(decolourizing agent), which removes thepurple from the cells of some species but

not from others.

4. The alcohol is rinsed off, & the slide isthen stained with safranin(basic dye) for

1 minute.The smear is washed again, blotted dry &examined microscopically.





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Gram staining results- Indicate type of stain used, the reaction, &

the morphology of the cells observed

- Eg.(a) Round (spherical), purple (or dark blue)

cells are reported as Gram positive

cocci (GPC)(b) Rod-shaped, purple (or dark purple) cells

are reported as Gram positive bacilli

(GPB)11/10/2014DNS 0303 Microbiology & Parasitology93




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Gram positive cocci (GPC)

Gram positive bacilli (GPB)

Gram negative cocci (GNC)

Gram negative bacilli (GNB)

The standard abbreviations for the 4 typesof Gram stain & morphologyare



Acid Fast Staining


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= Ziehl-Neelsen staining Mycobacteria have waxy coats on their cell

walls that preventthem taking in the dyefrom the Gram staining procedure

Acid fast stainingdetergents are appliedwhich remove this waxy coat

Bacteria are stained hot Carbol-Fuchsin(red dye which contains detergents)slide is

gently heated for several minutesAllbacteria are then stained red

Heatingenhances penetration & retentionof the dye


Acid Fast Staining

Acid Fast Staining (Cont )


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The bacteria are washed with acid alcohol, ade-colourizer, which removesthe red stainfrom bacteria that are not acid-fast.

The acid-fast microorganisms retain red

colour because the carbol-fuchsin is moresoluble in the cell wall lipids than in the acid-alcohol.

Then stained with methylene blue (bluedye). Those bacteria that retain the red dyefrom the original stain are known as acid-fastbacteria, all others go blue.


Acid Fast Staining (Cont.)



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Acid fast bacilli




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