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Diagnostic Microbiology

The practical classification scheme of medical microorganisms shown in table

(1) in which organisms are grouped according to a few shared characteristics.

Protozoa and Fungi can often be identified on morphological criteria alone.

Identification of the organism causing an infectious process is usually essential

for effective antimicrobial and supportive therapy. Microbiologic diagnosis involves:

1) microscopic appearance : Gram stain , acid fast stain.

2) macroscopic appearance :

a) cultivation and identification of the organism.

b) detection of microbial antigens.

c) detection of microbial DNA or RNA.

d) detection of an inflammatory or host immune response to the microorganism

(Figure 4.1).

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Table 1 : Simple classification of some cellular micro-organisms of medical

importance

Common group name Normal genus names

Eukaryotes

Protozoa

Sporozoa Plasmodium ,Isospora , Toxoplasma ,

Cryptosporidium

Flagellates Giardia ,Trichomonas ,Trypanosoma ,

Leishmania

Amoebae Entamoeba ,Naegleria , Acanthamoeba

Other Babesia ,Balantidium

Fungi

Mould-like Epidermophyton ,Trichophyton , Microsporum ,

Aspergillus

Yeast-like Candida

Dimorphic Histoplasma ,Blastomyces , Coccidiodes

True Yeast Cryptococcus

Prokaryotes

Bacteria

Filamentous bacteria Actinomyces ,Nocardia , Streptomyces , Mycobacterium

True bacteria'

Gram-positive bacilli Aerobes: Corynebacterium , Listeria , Bacillus

Anaerobes: Clostridium , Lactobacillus , Eubacterium

Gram-positive cocci Staphylococcus ,Sterptococcus , Enterococcus

Gram-negative cocci Aerobes:Neisseria

Anaerobes:Veillonella

Gram-negative bacilli Aerobes: Enterobacteria = Escherichia, Klebsiella ,Proteus

,Salmonella ,Shigella , Yersinia. Pseudomonads=

Pseudomonas ,Burkholderia , Stenotrophomonas

Parvobacteria –Haemophilus , Bordetella , Brucella

,Pasteurella

Anaerobes :Bacteroides ,Fusobacterium

Gram-negative vibrios Vibrio ,Spirillum , Campylobacter ,

And spirilla Helicobacter

Spirochaetes Borrelia ,Treponema , Brachyspira,

Leptospira

Mycoplasmas Mycoplasma ,Ureaplasma

Rickettsiae ,Chlamydia Rickettsia , Coxiella , Chlamydia

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1 – Microscopic appearance :-under the compound light microscope which include

a . Reaction with gram stain or acid fast stain.

b .Morphology and arrangement .

c . Capsulated or not .

d . Motile or not .

e . Spore forming or not .

Preparation of fixed smears:

A- From fluid material (e.g. broth culture, urine, sputum, pus, exudates,

….ect).

1- Sterilize the loop on Bunzen flame, and let it to cool.

2- Under aseptic technique, withdraw a loopful of the specimen and spread it on

the center of a clean slide to form a somewhat thick film of 1-2cm in diameter,

then resterilize the loop.

3- Allow the film to dry without heating (on air).

4- Fix the film on the slide by passing it three times through the Bunzenflame,

allow the slide to cool before staining.

B- From solid material (e.g. colonies on agar).

1- Sterilize the loop on Bunzen flame and let it to cool.

2- Place a loopful of a clean water (tap water can be used) on the center of a clean

slide.

3- By the resterilized loop, transfer a small portion of the colony to the water

drop, emulsify thoroughly and spread the mixture evenly on the slide to form a

thin film of 1-2 cm in diameter.

4- Dry and fix ( as mentioned above).

Aims of fixation

1- Kill the microorganism.

2- Make the M.O. more stuck to the slide surface.

3- Make the M.O. more permeable to the stain.

4- Prevent the M.O. from undergoing autolytic changes.

Gram stain:

Is one of the most important methods widely used in bacteriology.Bacteria

according to Gram stain fall into 2 categories:

1-Those retained the first dye (crystal violet) throughout the staining procedure are

known as "Gram Positive" bacteria,appear violet.

sknown as "Gram Negative" bacteria,appear red.Therefore it is possible to

differentiate between bacteria of the same morphology.

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Procedure:

1-flood the fixed slide with crystal violet (primary dye) for 1-2 min, then wash

with water.

2-apply grams iodine (mordant) for 1-2 min, then wash with water.

3-apply 95% ethyl alcohol (decolorizer) for 30 seconds then wash with water.

4-apply saffranin (counter stain) for 0.5-1 min, wash with water then dry on air.

5-examine under oil immersion lens.

Mechanism of staining by Gram stain:

The division of bacteria into Gram positive and Gram negative indicates a basic

chemical differences between them in the cell wall:

1-The cell wall of Gram negative bacteria have relatively little peptidoglycan and

mainly consist of lipid. Lipid is soluble in alcohol so alcohol will extract lipid

material from G-ve bacterial cell wall, the crystal violet-iodine complex flow out of

the cell leaving it colorless. So, when we apply the saffranin (counter stain), bacterial

cell will take this color and appear red in color. While in Gram +ve bacteria,

peptidoglycan comprises a major part of the cell wall, so it will be more rigid than G-

ve bacteria and less permeable for the crystal violet-iodine complex and will not leaks

out of the cell during the process of decolorizing and the cell wall will not affect by

alcohol because it is insoluble in alcohol.

2-The more acidic character of the protoplasm of G+ve bacteria which is enhanced by

treatment with iodine may partly explain their stronger retention of the basic dye.

Acid Fast stain (Ziehl-Neelsen stain):

It is special stain for Mycobacterium tuberculosis. This bacterium is stained with

dye carbolfuchsin, they resist the effect of strong decolorizing agents as acid alcohol

so appear as red (acid fast positive) ,while the background and other m.o. will not

resist acid alcohol so appear blue (acid fast negative) because stained by the counter

stain (methylene blue) .M. tuberculosis has a waxy component (mycolic acids) in

their cell wall that enable it to retain the primary stain. So all cells on the slide will be

decolorized except M. tuberculosis .

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2. Macroscopic appearance

a . Culture appearance :

Types of media:

Simple (basal) media:

It uses for cultivation of common m.o. but not for fastidious bacteria .e.g nutrient

broth, nutrient agar.

Complex media:

1- Enriched Media : It is simple media enriched with one of the following

substance (blood, serum, glucose,…) it used to cultivate fastidious m.o. e.g.

Neisseria gonorrhoeae, Haemophilus influenzae e.g (blood agar, Chocolate

agar).

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.2-Selective media

It is media favor the growth of certain bacteria and inhibit growth of other bacteria

because it contain inhibitory substance as antibiotics, dyes, chemicals, alteration of

PH. e.g. Mannitol salt agar : contain high conc. of NaCl (7.5%) that most bacteria

cannot grow in this conc. except Staphylococcusspp

MacConkey᾽s agar: contains bile salt that inhibit all kinds of bacteria except

Enterobacteriacea.

Salmonella shigella agar (SS) is selective for Salmonella and shigella spp.

3-differential media

Distinguish one m.o. type from another growing on the same media. It is used

to recognize certain spp .of bacteria either by:

1 -Fermentation or non fermentationof lactose on MacConkey᾽s agar (this

media contain neutral red as indicator which change in color during

fermentation , because the gas will be produced and decreased the pH.

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2-Hemolysis or no hemolysis on blood agar:

Beta hemolytic β (complete hemolysis ) e.gStreptococcus pyogenes

Alpha hemolytic α (incomplete hemolysis ) e.g Streptococcusviridans

Gamma hemolyticϒ ( no hemolysis ) e.g Enterococcus faecalis

Macroscopic characteristics of the microorganism in any culture

Size :Shape:Elevation:Surface structure:Edge:Color and

opacityConsistency:Emulsifinibilit

phenomenon : swarming (Proteus ), Medusa head (Bacillus

left: no lactose fermentation

right: lactose fermentation

MacConkey Agar

Swarming of Proteusmirabilis Medusa head of bacillus anthracis

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pigment:

a. exopigment : e.g Pseudomonas aeruginosa produce pyocyanine (green pigment)

and Pseudomonas fluorescens produce flourscine (blue pigment)

b. endopigment :

e.g Staphylococcus aureus (golden colonies ), Staphylococcus citrus(yellow

colonies) , Staphylococcus albus(white colonies ).

Odor : sweet odor (apple) such as Pseudomonas.

bad odor (fish) such as Proteus .

haemolysis in blood:as mentioned before

Methods of inoculation and isolation of pure culture

Mixed bacterial population; sputum, urine, pus, infected wound, abscess, ….ect.

Pure culture: a single kind of m.o. growing alone in a protected environment.

Bacterial colony: a mass composed of identical bacterial cells.

Pseudomonasaeruginosa Pseudomonas fluorescens

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Methods :

1- the streak plate method

2- the pour plate method

3- the serial dilution method

5- the micromanipulater technique

The streak plate method

(Quadrant steak)

Procedure:

1-sterilize the loop, take one loopful of the m.o.

2- streak one loopful of m.o. over area (A) lightly near edge of the plate, don’t

gouge into the medium

3- re-sterilize the loop and cool it, rotate the dish 90° while keeping the dish

closed, streak area (B) with several back and forth strokes, hitting the original

steak a few times.

4- flame the loop again. Rotate the dish 90° and streak area (C) several times,

hitting last area several times.

5- flame the loop, cool it, rotate the dish 90°, streak area (D) , contacting area (C)

several times and drag out the culture.

6- flame the loop, incubate the plate at 37° for 24 hrs.

7- a subculture is done after incubation by transferring one of the growing

colonies to a new sterile medium, follow the same technique mentioned above,

the new culture will be a pure one.

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B-Biochemical test

Single-enzyme tests:

Some enzymes are necessary for the bacterium's individual metabolism and some

facilitate the bacterium's ability to compete with other bacteria or establish an

infection. They can be performed on organisms grown in culture, and often provide

identification.

Catalase test: in differentiating between many gram-positive organisms;e.g.,

Staphylococci are catalase-positive, whereas Streptococci and Enterococci are

catalase-negative.

Oxidase test: in differentiating between groups of gram-negative bacteria.e.g.

Pseudomonas , Neissera are oxidase-positive.

Urease: The test helps to identify certain species of Enterobacteriaceae,

Corynebacterium and Helicobacter pylori.

Coagulase test: in differentiating Staphylococcus aureus (coagulase-positive)

from coagulase-negative Staphylococci.

Tests based on the presence of metabolic pathways, EPI 20:

These tests measure the presence of a metabolic pathway in a bacterial isolate,

rather than a single enzyme.

EPI20 (enzyme profile index) widely used manual system for the family

Enterobacteriaceae and other gram-negative bacteria makes use of twenty

microtubes containing substrates for various biochemical pathways. The test

substrates in the microtubes are inoculated with the bacterial isolate to be identified,

and, after five hours' incubation, the metabolic profile of the organism is constructed

from color changes in the microtubes. These color changes indicate the presence or

absence of the bacteria's ability to metabolize a particular substrate. The results are

compared with a data bank containing test results from known bacteria (Figure

4.10). The probability of a match between the test organism and known pathogens is

then calculated.

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C-Detection of Microbial DNA or RNA

A highly specific method of pathogen detection involves identification of its DNA

or RNA in a patient sample ,specially for m.o. that cannot be grown in vitro like

Mycobacterium leprosy and Treponema pallidium. Also they are useful in the

detection of organisms that require complex media or cell cultures and/or prolonged

incubation times. These methods like: direct hybridization, and amplification

methods using the polymerase chain reaction1 or one its variations.

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ANTIBIOTIC SENSITIVITY TESTING

Is a laboratory test use to measure the ability of an antimicrobial agent to kill or

inhibite the growth of m.o in vitro,and assist the clinicians in the choice of drug

for treatment of infections in vivo.According to this the m.o classified into:

Sensitive, Intermediate, Resistant.

There are 4 methods to perform the antibiotic sensitivity test :

1- disc diffusion

2- broth dillution

3- E- test

4- vitek 2 test

1-Disc agar diffusion (Kirby-Bauer) test

Dependent upon the inhibition of reproduction of a m.o on the surface of a solid

medium by an antimicrobial agents which diffuses into the medium from a filter

paper disc into the medium . If the organism is killed or inhibited by the

concentration of the antibiotic , there will be no growth in the immediate area

around the disc : this is called the zone of inhibition .

Materials and reagents

The media: Muller –Hinton agar or blood agar

Antimicrobial discs: commercially available disc are used

The sample: after isolation and identification of bacteria

Procedure

1- Preparethe inoculum : by bacterial inoculum in nutrient broth which is prepared

by Picking 3-5 isolated colonies from the plate

2-Adjust the turbidity tothesame as the McFarland No. 0.5standard.*

3-Dipping a sterile swab into the broth

4-Streak the swabonthe surface of theMueller-Hinton agaror nutrient agar (3 times

in 3 quadrants)

5-Leave 5-10 min to dry the surface of agar which allowing the bacteria to establish

themselves on the media.

6-Place the appropriate drug immpregnant disc on the surface of the inoculated agar

plate.

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7-Invert the plate and incubate them at 37ºC for (18-24 h).

8- Take the culture plate, place the metric ruler across the zone of inhibition, at the

widest diameter and measure from one one edge of the zone to the other edge.

The disc diameter will actually be part of the number . Zone diameter result

reported in milimeter , looked up on the chart and result reported as sensitive

(S), resistant (R) ,or intermediate (I).

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2-Dilution antibiotic sensitivity test

a laboratory test used when the phycision need to determine the minimum

concentration of antibiotic that required to control the infection .

Minimum Inhibition Concentration (MIC)

The lowest concentration of antimicrobial agent that inhibits bacterial growth/

multiplication

Minimum Bactericidal Concentration (MBC) or Minimum Lethal

Concentration (MLC)

3-E-test

is a well established AST method in microbiology laboratories around the world.

The E technique comprises a predefined gradient of antibiotic

concentrations on a plastic strips.

4-VITEK 2 system Reporting of Resistance

Is the most developed software system in this field, and is capable of

identifying even low-level resistance.