Microbiology Guide - Introduc FK-Unisba

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Microbiology could be defined as the study of organisms too small to be seen with the naked eye. Figure 1.1 shows the relative size of microbes

compared to other living things. However, the relatively recent discovery of bacteria of near 1 mm in size has made this definition somewhat

inaccurate and in the grand tradition of science, a new definition is in order.

The chart above shows how microorganisms are related.

The three most general groups into which the organisms

are placed are prokaryotes, eukaryotes, and non-living organisms.

We will consider microbiology to be the study of organisms that can exist as single cells, contain a nucleic acid genome for at least some part of their

life cycle, and are capable of replicating that genome. This broad description encompasses an understandably large group of organisms including

fungi, algae, protozoa and bacteria. This definition would also include viruses, which microbiology texts traditionally discuss along with living

organisms.

What is microbiology ?

Figure 1.1 The relative size of microbes. Though microbes are small, they nevertheless

span a large range of sizes from the smallest bacterial cells at ~0.15 µm to giant bacteria

larger than 700 µm. The viruses depicted at the far left of the scale are even smaller.




Microbiology also involves a collection of techniques to study and manipulate these small creatures. Because of their size, special instruments and

methods had to be developed to allow the performance of interpretable experiments on microorganisms. These methods are not restricted to

microbes alone, but have also found utility in working with populations of cells from higher organisms.

Microorganisms are everywhere, but why are they worth learning about? The short answer is that they affect your life in many different ways.

Before we begin our study of these creatures, we will first take a tour of some of their important habitats and point out why your existence depends

upon them. We will then briefly explore the history of microbiology.

If you ask the average person how microbes (or germs) impact their lives, they would immediately think of disease. This is not a silly v iew, aspathogenic microorganisms have greatly affected human populations throughout our existence. Until about 1930, microbes were the major cause of

death in humans, with infectious disease infant mortality rates above 50%. From today’s perspective this is a horrendous stat istic, over half of all

infants did not make it to adulthood! With the advent of antibiotics, vaccines and better water sanitation, humanity has reduced the impact

of pathogenicmicrobes, but they will always remain an important social concern. The discipline of microbiology emerged from the study of these

diseases and most advances in treating various ailments had their roots in this relatively young scien ce.

From the beginning of microbiology, significant resources have been spent to understand and fight disease -causing microorganisms. You may be

surprised to learn that only a small fraction of microbes are involved in disease, many other microbes actually enhance our well being.

In fact, like all other large organisms, humans are actually consortia of different organisms - there are more non-human cells in and on our bodiesthan there are human cells! Recent experiments, that have examined microorganisms inside our digestive tract by intensive sequencing experiments

have revealed many interesting findings. More than 80% of the microbes in our guts have not been cultured. In addition, the m icrobial flora of a

person is unique to that person, and there are differences based upon body type and genetic background. This has profound effects on physical well-

being of the individual.

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Microbiological investigation

METHODS OF BACTERIAL IDENTIFICATION

Macroscopic morphology (appearance of bacterial colonies on petri dish

Microscopic morphology (bacterial shape & arrangement under the microscope)

Physiological / biochemical characteristics (metabolism: aerobes vs anaerobes)

Chemical analysis (cell wall composition)

Serological analysis (antibodies)

Genetic & molecular analysis (DNA & rRNA sequence)







Identification plan for

genus Staphylococcus
















Gram Positive Organisms

Aerobic, Gram-positive cocci

Staphylococcus aureus

Staphylococcus epidermidis Staphylococcus sp. (Coagulase-negative)

Streptococcus pneumoniae (Viridans group)

Streptococcus agalactiae (group B)

Streptococcus pyogenes (group A)

Enterococcussp.

Aerobic, Gram-positive rods

Bacillus anthracis

Bacillus cereus Bifidobacterium bifidum

Lactobacillus sp.

Listeria monocytogenes

Nocardia sp.

Rhodococcus equi (coccobacillus)

Erysipelothrix rhusiopathiae

Corynebacterium diptheriae

Propionibacterium acnes

Anaerobic, Gram-positive rods

Actinomyces sp.

Clostridium botulinum

Clostridium difficile

Clostridium perfringens

Clostridium tetani

Mobiluncus sp. (gram-variable or gram-negative but has a gram-positive cell wall)

Anaerobic, Gram-positive cocci

Peptostreptococcus sp.




Gram Negative Organisms

Aerobic, Gram-negative cocci

Neisseria gonorrhoeae

Neisseria meningitidis

Moraxella catarrhalis

Anaerobic, Gram-negative cocci

Veillonella sp.

Aerobic, Gram-negative rods Fastidious, Gram-negative rods

o Actinobacillus actinomycetemcomitans

o Acinetobacter baumannii ( A. calcoaceticus)

o Bordetella pertussis

o Brucella sp.

o Campylobacter sp.

o Capnocytophaga sp.

o Cardiobacterium hominis

o Eikenella corrodens

o Francisella tularensis

o Haemophilus ducreyi

o Haemophilus influenzae

o Helicobacter pylori

o Kingella kingae

o Legionella pneumophila

o Pasteurella multocida

o Klebsiella granulomatis (formerly

calledCalymmatobacterium granulomatis (Gram

negative rod) Enterobacteriaceae (glucose and lactose fermenting

Gram-negative rods)

o Citrobacter sp.

o Enterobacter sp.

o Escherichia coli

o Klebsiella pneumoniae

Fermenting glucose but NOT lactose; Gram-negative rods

o Proteus sp.

o Salmonella enteriditis

o Salmonella typhi

o Shigella sp.

o Serratia marcescens

o Yersinia enterocolitica

o Yersinia pestis Oxidase-positive, glucose-fermenting Gram-negative rods

o Aeromonas sp.

o Plesiomonas shigelloides

o Vibrio cholerae

o Vibrio parahaemolyticus

o Vibrio vulnificus

Glucose-nonfermenting, Gram-negative rods

o Acinetobacter sp.

o Flavobacterium sp.

o Pseudomonas aeruginosa

o Burkholderia cepacia

o Burkholderia pseudomallei

o Xanthomonas maltophilia or Stenotrophomonas

maltophila

Anaerobic, Gram-negative rods

Bacteroides fragilis

Bacteroides sp.

Prevotella sp.

Fusobacteriumsp.

Gram-negative spiral

Spirillum minus (minor )-




Gram stain




(purple dye)

(mordant)

alcohol

(safranin as counterstain)




Acid fast stain

(Ziehl Neelsen stain)

Mycobacterium tuberculosis bacteria (Magnified 1000X).

Acid fast organisms stain red.

Non acid fast organisms and tissue cells stain blue.




S t i




Spore stain

bacillus subtilis

Spore Stain of Bacillus megaterium The cells in this figure were stained with malachite green to

stain endospores and the vegetative cells were stained with

safranin. The top arrow is pointing to a free endospore, the

middle arrow is pointing to a red vegetative cell which doesnot have an endospore, and the lower arrow is pointing to a

vegetative cell which still has the endospore with in it. The

red outline of the cell is still visible but the endospore takes

up most of the cell space.

The Gram-positive Clostridium subterminale bacteria, which

had been cultivated on a blood agar plate (BAP)




smear with malachite green

heat over the flame for 3 min.

Keep the smear covered with the

dye and don’t allow to boil



















Escherichia coli (Gram-negative rods)

Klebsiella pneumoniae & Staphylococcus aureus

Magnification: 1000×

Gram-negative rods and gram-positive cocci

(Gram-positive rods)


Moraxella catarrhalis


Gram-negative diplococci




Most isolation media are a combination of two or more types listed above.

Name of

MediaType Use in Medical Lab Ingredients Growth Allowed Growth Inhibited

Trypticase

Soy

Agar/Broth

TSA, TSB

General

All PurposeBasic nutrients Most organisms

Fastidious

organisms such

as Streptococcus

Blood Agar

BAP

Enrichment

Differential

1) To grow fastidious bacteria.

2) To differentiate between

bacteria based on hemolysis.

Ex/ Streptococcus

Beef heart

(enrichment)

Sheep RBCs

(differential)

Most organisms

including

Streptococcus

Few or none

MacConkey

Agar

MAC

Selective

Differential

1) To select for Gram negative

enteric bateria.


Gram negative enteric

bacteria based on lactose

fermentation.

Ex/ Escherichia coli

Bile Salts (Selective)

Crystal Violet

(Selective)

Lactose (differential)

Neutral Red (indicator)

Gram negative

coliforms, other

Gram negative

Gram positive

MannitolSalt Agar

MSA

Selective

Differential

1) To select for salt tolerant

Gram positive bacteria.


salt tolerant bacteria based on

mannitol fermentation.

Ex/ Staphylococcus aureus

7.5% Salt (selective)Mannitol (differential)

Phenol red (indicator)

Staphylococcus,Bacillus, other

Gram positive

Most other

bacteria

Microbiology agar plates




Chocolate agar (CA)Use : For the isolation and cultivation of a variety of fastidious microorganism.

CHOC is an enriched medium supplemented with cofactor, which provides NAD

to facilitate the growth of Haemophilus influenzae, Neisseria gonorrhoeae and

Neisseria meningitidis. Heated sheep blood is added to give the medium its “chocolate” appearance.

Thayer-Martin agar (TMA)Use : To select for fastidious organisms, such as N. gonorrhoeae, in patient samples

containing large numbers of normal flora, such as in the female genital tract

Microbiology agar plates

(media for cultured bacteria)




MacConkey agar (MC)Use : For the selective isolation, cultivation and differentiation of coliformsand enteric pathogens

based on the ability to ferment lactose. Lactose –fermening organisms appear as red to pink colonies.

Lactose-nonfermenting organisms appear as colorless or transparent colonies

Blood agar (BA)Use : For the isolation, cultivation and detection of hemolytic activity of

streptococci, pneumococci and other particular fastidious microorganisms




Salmonella-Shigella agar (SS)Use : For the selective isolation and differentiation of pathogenic enteric bacilli,

especially those belonging to the genus Salmonella. This media is notrecommended

for the primary isolation of Shigella species. Lactose-fermenting bacteria such as Escherichia coli or Klebsiella pneumoniae appear as small pink or red colonies.

Lactose-nonfermenting bacteria such as Salmonella species, Proteusspecies and

Shigella species appear as colorless colonies. Production of H2S bySalmonella species

turns the center the colonies black.

Eosin Methylene Blue agar (EMB)Use : For the isolation, cultivation and differentiation of Gram-negative enteric bacteria

based on lactose fermentation. Bacteria that ferment lactose, especially the coliform

bacterium Escherichia coli , Appear as colonies with green metallic sheen or blue –black to

brown color. Bacteria that do not ferment lactose appear as colorless or transparent light

purple colonies




Thiosulfate Citrate Bile Salt Sucrose agar (TCBS)Use : For the selective isolation of Vibrio cholerae and Vibrio parahaemolyticus

from a variety of clinical specimens and in epidemiological investigations.

Sabouraud Dextrose agar (SDA)Use : For the cultivation of pathogenic and nonpathogenic fungi, especially

dermatophytes. The medium may be made more selective for fungi by the addition

of specific antibiotics such as chloramphenicol. For the cultivation of yeast and

filamentous fungi.




Tryptic Soy agar (TSA)Use : Cultivation on non-fastidious bacteria

Mannitol Salt agar (MSA)Use : Selects for Staphylococci, which grow at high salt concentrations,

differentiates Staphylococcus aureus from other Staphylococci

Staphylococcusaureus

d




Lowenstein-Jensen Media Agar

for Mycobacterium

Mycobacterium tuberculosis colonies

Different mycobacteria species grown on TB-MediumBase according to Löwenstein- Jensen




Mueller-Hinton agar (MHA)Use : For antimicrobial susceptibility testing of a variety of

nonfastidious, rapidly-growing microorganisms.




Spirit Blue agarUse : This agar is used to identify organisms that are

capable of producing the enzyme lipase.

This enzyme is secreted and hydrolyzestriglycerides to glycerol and three long chain fattyacids. These compounds are small enough to passthrough the bacterial cell wall. Glycerol can beconverted into a glycolysis intermediate. The fattyacids can be catabolized and their fragments caneventually enter the Kreb’s cycle. Spirit blue agar

contains an emulsion of olive oil and spirit blue dye.Bacteria that produce lipase will hydrolyze the oliveoil and produce a halo around the bacterial growth.The Gram-positive rod, Bacillus subtilis is lipasepositive (pictured on the right) The plate pictured onthe left is lipase negative.

Hemolytic bacteria




Hemolysis

Alpha hemolysis: this is the partial destruction of red blood cells, and often has a greenish hue to it rather than an

actual clearing around the colonies,

Beta hemolysis: which is the complete destruction of red blood cells, usually, if you can see a finger or some other

object through the clearing on the plate

Gamma hemolysis: no hemolysis

Hemolytic bacteria

β α ϒ

Hemolysis




Hemolysis







Biochemical identification




Tests used to identify

Gram Positive Bacteria

Catalase Test

Mannitol Salt Agar (MSA)

Blood Agar Plates (BAP)

Motility Agar

Coagulase TestTaxos P (optochin sensitivity testing)

Taxos A (bacitracin sensitivity testing)

CAMP Test

Bile Esculin Agar

Nitrate Broth

Spirit Blue agar

Starch hydrolysis test

Tests used to identify

Gram Negative Bacteria

Oxidase Test

Sugar (eg glucose) broth with Durham tubes

Methyl Red / Voges-Proskauer (MR/VP)

Kliger’s Iron Agar (KIA)

Nitrate BrothMotility Agar

MacConkey agar

Simmon’s Citrate Agar

Urease test

Sulfur Indole Motility Media (SIM)

The Controls That Were Performed




The Controls That Were Performed

For The Various Tests

Urease test




Indole Testndole tests looks for the presence orabsence of tryptophanase enzyme

production of the bacteria. If the enzyme ispresent,it will degrade the aminoacid

tryptophan in the media and will produce

Indole,ammonia and pyruvic acid. Indole will

react with Kovac’s reagent to produce acherry red complex,which indicates a

positive indole test. The absence of red color

is indicative of tryptophan hydrolysis due tothe lack of tryptophanse enzyme

Certain bacteria produce the

enzyme urease during its

metabolism process and that

will break down the urea in

the medium to ammonia and

carbon dioxide creates an

alkaline environment that

causes the phenol red to turn

to deep pink. This is a positive

reaction for the presence of

urease. Failure of deep pink

color to develop is evidence of

a negative reaction

Strong

Urease

Uninoculated

Tube

Negative

Urease

TSI (triple sugar iron)




TSI (triple sugar iron)







Result (slant/butt) Symbol Interpretation

1 Red/Yellow K/A Glucose fermentation only, peptone catabolized.

2 Yellow/Yellow A/A Glucose and lactose and/or sucrose fermentation.

3 Red/Red K/K No fermentation, Peptone catabolized.

4 Yellow/Yellow with bubbles A/A,GGlucose and lactose and/or sucrose fermentation, Gas

produced.

5 Red/Yellow with bubbles K/A,G Glucose fermentation only, Gas produced.

6 Red/Yellow with bubbles and black precipitate K/A,G,H2S Glucose fermentation only, Gas produced, H2S produced.

7 Yellow/Yellow with bubbles and black precipitate A/A,G,H2SGlucose and lactose and/or sucrose fermentation, Gas

produced, H2S produced.

8 Red/Yellow with black precipitate K/A,H2S Glucose fermentation only, H2S produced.

9 Yellow/Yellow with black precipitate A/A,H2SGlucose and lactose and/or sucrose fermentation, H2S

produced.




DNAse Test




Note there is breakdown of the DNA in the agar. There is a clear zone (arrow) around the bacterial growth where there is no longer any

DNA left in the agar to precipitate out of solution after the HCl was added.

General

Many bacteria have enzymes that break down nucleic acids. The bacteria can then use the resulting nucleotides to build up their own nucleic acids. DNase is suchan enzyme, which thus hydrolyzes DNA. Existence of DNase is characteristic for certain species or strains of bacteria and can be used for typing.

Method

Presence of DNase can be determined by cultivation on an agar plate, which contains DNA. If the bacterium has DNase and if the bacteria are allowed to grow over

night, the DNA will be hydrolyzed into the constituting nucleotides. Diluted hydrochloric acid (HCl) is then poured onto the plate and there will be a clear zone close

to the colonies or the streak, because individual nucleotides are soluble in diluted HCl, but not DNA, which precipitates in the rest of the plate.

DNAse Test




CITRATE TEST

The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energysource. Simmon's media contains bromthymol blue, a pH indicator with a range of 6.0 to 7.6. Bromthymol blue is yellow at acidic pH's

(around 6), and gradually changes to blue at more alkaline pH's (around 7.6). Uninoculated Simmon's citrate agar has a pH of 6.9, so it is

an intermediate green color. Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate).

Enterobacter and Klebsiella are citrate positive while E.coli is negative.

CATALASE TEST (OXIDATIVE TEST)




CATALASE TEST (OXIDATIVE TEST)

Catalase positive Catalase negative

Catalase production and activity can

be detected by adding the substrate

H2O2 to an appropriately incubated(18- to 24-hour) tryptic soy agar slant

culture. Organisms which produce

the enzyme break down the

hydrogen peroxide, and the resulting

O2 production produces bubbles in

the reagent drop, indicating a

positive test. Organisms lacking the

cytochrome system also lack the

catalase enzyme and are unable to

break down hydrogen peroxide, into

O2 and water and are catalase

negative




COAGULASE

TEST

Staphylococcus

aureus

Staphylococcus

epidermidis

Gelatin hydrolysis tes




Gelatin hydrolysis tes

It is broken down

by gelatinase into smallerpolypeptides, peptones and amino

acids that can cross the cell

membrane and be utilised by the

organism.

when gelatin is broken down via

hydrolysis, it cannotsolidify

anymore, the areas of solid gelatin

media where the organsim grows,will turn into liquid. Even if you

refrigerate this medium, the

media will remains liquid.

Bacillus subtilisis able to produce

proteolytic exoenzyme gelatinase

to give the (+) result.

METHYL RED TEST




This test detects the ability of

microorganism to ferment glucose

and to produce acidic end products.

Enteric organism produces pyruvic

acid from glucose metabolism.

Some enteric will thn use the Mixed

acid pathway to metabolize pyruvicacid to other acidic products such as

lactic acid,acetic acid and formic

acids. This will reduce the pH of the

media. Methyl red is a pH indicator

which is red at the acidic pH (below

4.4) and yellow at alkaline pH(

above 7). The formation of red color

after the addition of Methyl red

reagent indicates the accumulation

of acidic end products in the

medium and is an indicative of

positive test

S tibilit t t




Susceptibility test

Depicted above are two methods for

determining antimicrobial susceptibility of




For this example the MIC for erythromycin is 0.19 µg/ml which is considered to be susceptible based on CLSI breakpoints. Consult the latest CLSI

manual* for interpretation of MICs for Streptococcusspp. Beta-hemolytic Group (2010 breakpoints): Clindamycin: ≤0.25 μg/ml = susceptible, 0.5

μg/ml = intermediate, and ≥1.0 μg/ml = resistant; and for Erythromycin: ≤0.25 μg/ml = susceptible, 0.5 μg/ml = intermediate, and ≥1.0 μg/ml =

resistant. The two disks on the right of the agar plate are erythromycin (E) and clindamycin (DA). There are criteria for the zones of inhibition of

growth that determine whether or not the bacteria are susceptible or resistant. In the test shown above, the bacterium is susceptible to

erythromycin (the zone is ≥21 mm) and susceptible to clindamycin (zone is ≥19 mm). The disk test is perfectly satisfactory for determining the

antimicrobial susceptibility of GBS. Interpret according to CLSI guidelines for Streptococcus spp. Beta-hemolytic Group (2010 breakpoints for disk-diffusion: for clindamycin: ≥19 mm = susceptible, 16–18 mm = intermediate, and ≤15 mm = resistant; for erythromycin: ≥21 mm = susceptible,

16–20 mm = intermediate, and ≤15 = resistant.

*Clinical and Laboratory Standards Institute. Performance standard for antimicrobial susceptibility testing, M100-S20 (2010), M-2 and M-7, Wayne, PA.

group B Streptococcus (GBS). It is especially

important that the microbiologist determine

the susceptibility of GBS to erythromycin and

clindamycin as these two drugs are used as

substitutes if the patient cannot be treated

with penicillin. The long strip on the left iscalled the E-testTM. This picture shows the

drug erythromycin (EM). In this test, the strip

contains a gradation of antibiotic with the

strongest being at the top of the strip and the

weakest at the bottom. The minimum

inhibitory concentration (MIC) of antibiotic

that will inhibit the bacteria is determined by

where the growth of the bacteria starts, or the

area of the growth where the ellipse growthmeets the strip.

T P ( t hi iti it t ti )




Taxos A (bacitracin sensitivity testing)

This is a differential test used to distinguish between organismssensitive to the antibiotic bacitracin and those not. Bacitracin is apeptide antibiotic produced by Bacillus subtilis. It inhibits cell wall

synthesis and disrupts the cell membrane. This test is commonly used

to distinguish between the-hemolytic streptococci: Streptococcus

agalactiae (bacitracin resistant) and Streptococcus pyogenes (bacitracin sensitive). The plate below was streakedwith Streptococcus pyogenes; notice the large zone of inhibitionsurrounding the disk.

Taxos P (optochin sensitivity testing)

This is a differential test used to distinguish between organisms

sensitive to the antibiotic optochin and those not. This test is used to

distinguishStreptococcus pneumoniae (optochin sensitive (pictured

on the right below)) from other a-hemolytic streptococci (optochin

resistant (Streptococcus mitis is pictured on the left below).

Carbohydrate

f t ti




The identification of some bacteria is aided by determining what nutrients the bacteria can

utilize and what end products will be produced in the process. These characteristics are

controlled by the enzymes which the bacteria produce. Because the type of enzyme(s) bacteria

produce is genetically controlled, the pattern of sugars fermented may be unique to a

particular species or strain. Fermentation products are usually acid (lactic acid, acetic acid etc.),neutral (ethyl alcohol etc.), or gases (carbon dioxide, hyrogen, etc.).

A bright yellow color indicates the production of enough acid products from fermentation of

the sugar to drop the pH to 6.9 or less. Production of gas is determined with a Durham tube, a

small inverted vial filled with the carbohydrate fermentation broth. If gas is produced during

fermentation of the sugar, it is trapped at the top of the Durham tube and appears as a bubble.

fermentation

Table of durham fermentation




Table of durham fermentation




If the laboratory is not able to identify group B streptococci (GBS) by the Lancefield grouping procedure, there are other microbiologic tests that

can be used to identify GBS. This picture shows one of these tests. It is called the CAMP test. CAMP is an acronym for the authors of this test

(Christie, Atkinson, Munch, Peterson). The CAMP test takes advantage of the capacity of GBS to produce this CAMP factor; most other hemolytic

streptococci do not produce CAMP factor. This picture shows the group B Streptococcus (on the right) and a group A Streptococcus (GAS) (on the

left). Down the middle we have inoculated the plate with a Staphylococcus aureus strain (vertical streak). We then inoculated the GBS (on right)

and GAS (on left) perpendicular to the Staphylococcus streak. We inoculated the agar plate so as not to touch the two different organisms(Staphylococcusand Streptococcus) but to come close to each other. TheStaphylococcus is used because it produces a lysin that only partially lyses

the red blood cells (called beta-lysin). The CAMP factor reacts with the partially lysed area of the blood agar plate to enhance the hemolytic

activity. Note the arrowhead shape of the zone of enhanced hemolytic activity by the GBS near the Staphylococcus streak (on right) but not by the

GAS (on left). This means that the bacterium on the right is GBS because it is producing a CAMP factor.

CAMP

TEST

Oxidative-Fermentative Metabolism ecoli, eaerogenes (controls included) OF test

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ecoli, eaerogenes (controls included) OF test

How do we determine if type of metabolism is

fermentative or oxidative? we will test with a

glucose OF oxidative- fermentative agar.

If agar turns yellow, acid is produced. If agar

turns green, no acid is produced. Motility of the

bacteria can also be determined by the presence of

turbidity (cloudiness)

OXIDATIVE metabolism may or may not cause an

acid to be produced for aerobic conditions. No acid

will be produced for anaerobic conditions.

FERMENTATIVE metabolism will cause an acid to

be produced for aerboic and anaerobic conditions.

Oxidative-Fermentative Metabolism OF test(yellow-acid, green-no acid)






(y , g )

Protein Catabolism (Proteolysis)

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Casein(a protein) is broken down by protease into peptones and amino acids. During the degradation process, polypeptide bonds are broken.

Once the bonds are broken, amino acids are produced. A clear zonesurrounding streak line of agar

indicates a (+) result.

Voges Proskauer Test




This test determines the ability of

microorganism to ferment glucose. The

end products of glucose

metabolism,pyruvic acid, is further

metabolized by using Butylene glucol

pathway to produce neutral end such as

acetoin and 2,3 butanediol. When

Barrit’s reagent A ( 40% KOH) and

Barrit’s reagent B(5% solution of alpha

naphthol) is added it will detect the

presence of acetoin,the precursor in the

2,3- butanediol synthesis. Acetoin in the

presence of Oxygen and Barrit’s reagent

is oxidized to diacetyl. in the presence of

alpha naphthol catalyst. Diacetyl then

reacts with guanidine components of

peptone to produce a cherry red color

Nitrate testThis is a differential medium. It is used to determine if anorganism is capable of reducing nitrate (NO3

-) to nitrite(NO2

-) or other nitrogenous compounds via the action of theenzyme nitratase (also called nitrate reductase). This test isi t t i th id tifi ti f b th G iti d




important in the identification of both Gram-positive andGram-negative species.

After incubation, these tubes are first inspected for thepresence of gas in the Durham tube. In the case ofnonfermenters, this is indicative of reduction of nitrate to

nitrogen gas. However, in many cases gas is produced byfermentation and further testing is necessary to determine ifreduction of nitrate has occurred. This further testingincludes the addition of sulfanilic acid (often called nitrate I)and dimethyl-alpha-napthalamine (nitrate II). If nitrite ispresent in the media, then it will react with nitrate I andnitrate II to form a red compound. This is considered apositive result. If no red color forms upon addition of nitrate Iand II, this indicates that either the NO3

- has not beenconverted to NO2

- (a negative result), or that NO3- was

converted to NO2- and then immediately reduced to some

other, undetectable form of nitrogen (also a positive result).

In order to determine which of the preceding is the case,elemental zinc is added to the broth. Zinc will convert anyremaining NO3

- to NO2- thus allowing nitrate I and nitrate II

to react with the NO2- and form the red pigment (a verified

negative result). If no color change occurs upon addition ofzinc then this means that the NO3

- was converted to NO2-

and then was converted to some other undetectable formof nitrogen (a positive result).

If the nitrate broth turns red (tubes pictured in the center)after nitrate I and nitrate II are added, this color indicates a

positive result. If instead, the tube turns red (tube picturedon the left) after the addition of Zn, this indicates a negativeresult. If there is no color change in the tube after theaddition of nitrate I and nitrate II, the result is uncertain. Ifthe tube is colorless (picture on the right) after the additionof Zn this indicates a positive test.

Bile esculin test




Bile Esculin Agar is recommended

for the presumptive isolation and

identification of group Dstreptococci.

Bile Esculin Agar contains esculin,

ferric citrate and 4% oxgall. This

concentration of oxgall will inhibit

practically all strains ofstreptococci

other than group D. If an organism

growing on the medium hydrolyzes

esculin, a glycone is formed. This

compound will react with the ferric

ions in the medium forming a black

coloration in the medium.

Bile solubility test




Use : To differentiates Streptococcus

pneumoniae from other α-hemolytic

streptococci by demonstrating itssusceptibility to lysis in the presence

of bile.

When a bile salt such as

desoxycholate is added directly

to Streptococcus pneumoniae growing

on an agar plate or in a broth culture

the bacteria will lyse and the areabecome clear. Other alpha-hemolytic

streptococci are resistant to (not

lysed by) bile and will stay visible or

turbid (cloudy)

This test is used to identify bacteria that can

Starch hydrolysis test




This test is used to identify bacteria that canhydrolyze starch (amylose and amylopectin) using

the enzymes -amylase and oligo-1,6-glucosidase.Often used to differentiate species from thegenera Clostridium and Bacillus. Because of thelarge size of amylose and amylopectin molecules,

these organisms can not pass through the bacterialcell wall. In order to use these starches as a carbon

source, bacteria must secrete-amylase and oligo-1,6-glucosidase into the extracellular space. Theseenzymes break the starch molecules into smallerglucose subunits which can then enter directly intothe glycolytic pathway. In order to interpret theresults of the starch hydrolysis test, iodine must beadded to the agar. The iodine reacts with the starchto form a dark brown color. Thus, hydrolysis of the

starch will create a clear zone around the bacterialgrowth.Bacillus subtilis is positive for starchhydrolysis (pictured on the left). The organism shownon the right is negative for starch hydrolysis.

Litmus milk test




Litmus milk

1 control, 2 pink = acid, 3 white = reduction, 4 & 5 stormy fermentation, 6 blue = alkaline

Use: To differentiate bacteria based on various reactions that occur in skim milk suplemented with a

litmus pH indicator.

Principle: Milk is a complex nutritional source that contains proteins (mainly casein) in an aqueous

solution of lactose and minerals. Bacterial enzymes alter the media and may bring about variouschanges. Litmus is added to the medium to detect pH changes that may occur as a result of these

enzymatic reactions. Above a pH of 8.3 litmus is blue, while below a pH of 4.5 litmus is red.

Microbiology : Biochemical Tests (Review)




Exoenzymes

Test Medium Substrate Product(s) Reagent Result

Amylase starch agar starch glucose, maltose, dextrins iodine added after incubation + = colorless zone around organism after iodine

Caseinase skim milk agar casein amino acids, peptides

+ = clear zone around organism

DNase DNase agar ds DNA nucleotides methyl green in agar (boundto DNA)

+ = colorless zone around organism

Lipase spirit blue agar lipid(triglyceride)

glycerol, fatty acidsspirit blue in agar

+ = blue/clear zone around organisms, blue ppt.in organisms

Selective and Differential Media

Test Medium SubstrateSelectiveIngredient

pHindicator

Result + organisms

EMB agar – selective for

gm - & differential forlactose fermentation

EosinMethyleneBlue (EMB)agar

LactoseEosin +Methylene Blue

Growth = gm -;Dye accumulation (color) in organism

= lactose fermentation:Metallic greenFish eyes (pink with purple center)

E. coliE. aerogenes

MacConkey’s agar – selective for gm - &differential for lactosefermentation

MacConkey’s agar

LactoseCrystal violet +bile salts

Neutral redin agar

Growth = gm -;Pink ppt. in organism = lactose fermentation

E. coli;E. aerogenes

Mannitol Salt Agar(MSA) – selective for staph& differential for mannitolfermentation

MSA Mannitol 7.5% NaClPhenol redin agar

Growth = salt tolerant (staphylococci)

Acidic (yellow) media = mannitol fermentation

S. epidermidis& S. aureus; S. aureus

IMViC

Test Media Substrate Product(s) Reagents pH indicator + appearance + organisms

Indole Tryptic Soy Broth (TSB) Tryptophan Indole Kovac’s layered on top Red at surface E. coliMethyl Red MRVP broth Glucose Organic acids Methyl red (Methyl red ) Red throughout E. coli Voges-Proskauer MRVP broth Glucose Non-acids VP-A & VP-B (Barritt’s) Red throughout E. aerogenes




Citrate Utilization Simmons’ citrate agar Citrate Na2CO3 Bromthymolblue in agar

Royal blue E. aerogenes

TSI (Triple sugar iron)

Test Media Substrate Product(s) Reagent pH indicator + appearance + organisms

H2S Gas TSI agar(stab & drag)

Cysteine H2S Iron in media Black ppt. when H2S reacts with ironto form iron sulfide

S. typhimuriumP. vulgaris

CarbohydrateFermentation

TSI agar(stab & drag)

Glucose, lactose,sucrose

Organic acids Phenol red inagar

Media: yellow = acidic (A);red = alkaline (K);

Record reactions as “slant”/”butt”:

K/A=glucose only, A/A=sucrose &/or lactose +/-glucoseK/K=none fermentedBlack butt = acidic

Carbohydrate Fermentation Series

Test Media Substrate Product(s) pH indicator + appearanceCarbohydrate Fermentation Carbohydrate brothwith Durham tubes

Glucose, lactose, or sucrose Organic acids Phenol red in broth Acidic (yellow) media

Gas Production CO2 Bubble in Durham tube

Test Media Substrate Product(s) pH indicator + appearance + organisms

Urease (Christensen’s) urea agar Urea Ammonia, CO2 Phenol red in agar pink (alkaline) media ProteusLitmus Litmus milk Casein, lactose Various Litmus in media pink = acid; sugar fermentation

hard curd = acidsoft curd = rennin clots caseinblue = alkaline; casein breakdownbrown = further casein breakdownwhite = litmus reduction

Test Substrate Product(s) Reagent + appearance + organisms

Catalase Hydrogen peroxide(H2O2)

Water, oxygen Bubbles Staphylococci

Coagulase Fibrinogen Clumping factor-fibrinogen complex Staphyloside latex beads Blue ppt. (clumping) S. aureus

(Cytochrome)O id

Cytochrome c Oxidized cytochrome c Phenylenediamine Blue color P. aeruginosa




Oxidase

Bile Solubility Tris buffer + deoxycholate Clearing (lysis of cells) S. pneumoniae

Hemolysis

Test Media Substrate Results + appearance + organisms

*Alpha Blood agar (5% SB) RBCs & hemoglobin (Hb) Hemolysis; partial digestion of Hb Green/brown zonearound/beneath organisms

Strep. pneumoniaeE.(S) faecalisStaph. epidermidis

**Beta Blood agar (5% SB) RBCs & hemoglobin (Hb) Hemolysis; complete digestion of Hb Clear zone around organisms Strep. pyogenes,E.(S) faeciumStaph. aureus

Gamma Blood agar (5% SB) RBCs & hemoglobin (Hb) No hemolysis No clearing aroundorganisms

* Alpha-hemolytic Strep differentiated by optochin sensitivity (S. pneumoniae)

**Beta- hemolytic Strep differentiated by bacitracin sensitivity (S. pyogenes)

Test Media Substrate Product(s) Reagent Appearance

Nitrate Reduction Nitrate broth Nitrate Nitrite;N2/ammonia

Nitrate A & B, zincadded after incubation

1. Add nitrate A & B:Red color = nitrate reductase 2. If colorless after nitrate A & B add zinc:Still colorless after zinc = both nitrate an d nitrite reductase If red after zinc = neither nitrate or nitrite reductase

Nitrate reductase Nitrite reductaseNitrate (NO3) Nitrite (NO2) N2 (gas) /NH3 (ammonia)

Nitrate A & B+ nitrate = colorless

Zincreduces nitrate to nitrite Nitrate A & B

+ nitrite = redNitrate A & B

+ N2 (gas) or NH3 (ammonia) = colorless




Bacterial Infection (Secondary Infection)

(sign / symptoms)

(Etiologic agent)

(Site of Infection)




(Site of Infection)

CLINICAL SPECIMENS

(kind of specimens)

GRAM POSITIVE COCCI GRAM NEGATIVE ROD

Blood agar Mac Conkey agar

(Measures of colony, Reaction on blood agar) (Morphology of colony, Reaction on M Conkey agar)

Lactose fermenter Non-Lactose fermenter

(Escherichia coli, (Salmonella thypi,

Klebsiella pneumoniaea-Enterobacter) Shigella flexneri)

Catalase

Catalase (-) Catalase (+)

(Streptococcus sp.) (Staphylococcus sp.)

S. Pneumoniae (Optochin +, Inulin +) S. aureus (Coagulase +)

S. Viridans S. epidermidis (Novobiocin +)

S. Hemolyticus S. saprophyticus

KIA,

MIU,

Citrate




Gram positive cocci

A Gram stain of Staphylococcus aureus (Gram positive cocci, purple).

Sheep blood agar:

Beta-hemolytic colonies indicate Staphylococcus aureus

Mannitol Salt Agar

Yellow colonies

Differential test for mannitol fermentors and non-fermentators;

S. aureus ferments mannitol.

Catalase test

Positive

DNA-se test

Positive

Coagulase test

Positive

Coagulase test differentiates S. aureus, from other

staphylococcus species

such as S. epidermidis present in the normal flora.

Antibiotic Susceptibility Tests

Vancomycin, Amoxicillin with clavulanic acid, Cloxacillin,

Ampicillin, Cephalosporon

grapelike cluster of

stphylococcus




Staphylococcus is a very well known genus of bacteria. Colonies are "gold," or yellow on

sheep blood agar solid media, hence the name. A common pathogen, boils, acne, wound

infections, food poisoning are among a host of conditions caused by this organism. The

organism is both pathogenic and invasive. It produces leukotoxin which can kill white

blood cells and a wide variety of other toxins. S. aureus is quite pyogenic and in decades

past was named Staphylococcus pyogenes, however that specific name is currently

applied to one GPC, Streptococcus pyogenes.

S. aureus on blood agar plate

S. aureus on mannitol agar plate




DNA-se test

Enterobacteriaceae




Gram negative - rod

McConkey agar

Lactose fermenter, mucous

Indole test

Positive

Methyl red test

Positive

Voges-Proskauer reaction test

Positive

Citrate utilization test

Positive

Urease test

Positive

Antibiotic Susceptibility Tests

Penicillin, Cephalosporin, Aminoglycoside,

Cephalosporin (e.g. Cefotaxime)

This inoculated MacConkey agar culture plate cultivated colonial growthof Gram-negative, small rod-shaped and facultatively anaerobic

String test of a Klebsiella pneumoniae isolatedemonstrating hypermucoviscosity




Pneumonia caused by Klebsiella pneumoniae.

K. pneumoniae can cause the disease Klebsiella pneumonia. They causedestructive changes to human lungs inflammation and hemorrhage with cell death(necrosis) that sometimes produces a thick, bloody, mucoid sputum (currant jellysputum). Typically these bacteria gain access after a person aspirates

colonizing oropharyngeal microbes into the lower respiratory tract.

Gram Stain of Sputum Specimen

Showing Capsules Surrounding a

Gram Ne ative Bacillus

Cut surface of normal lung

Klebsiella pneumoniae

biochemical test :

Indole positive

Urease positive

Citrate positive

Urease

test

http://en.wikipedia.org/wiki/Klebsiella_pneumonia

http://en.wikipedia.org/wiki/Inflammation

http://en.wikipedia.org/wiki/Hemorrhage

http://en.wikipedia.org/wiki/Necrosis

http://en.wikipedia.org/wiki/Oropharyngeal

http://en.wikipedia.org/wiki/Microbes

http://en.wikipedia.org/wiki/Microbes

http://en.wikipedia.org/wiki/Oropharyngeal

http://en.wikipedia.org/wiki/Necrosis

http://en.wikipedia.org/wiki/Hemorrhage

http://en.wikipedia.org/wiki/Inflammation

http://en.wikipedia.org/wiki/Klebsiella_pneumonia




Tripple Sugar Iron Medium (TSI medium)

Yellow/Yellow (acidic)

with bubblesA/A,G

Glucose and lactose and/or

sucrose fermentation, Gas

produced.

Citrate positive

Voges-Proskaueur positive

TSI : yellow/yellow, gas (A/A,G)

uninoculated A/A,G

Positive

citrate

Negative

citrate

Indole test

References :




References :

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education

Alfred.E.Brown. (2007). Bensons’s microbiological applications: laboratory manual in general microbiology. (10th ed.). New

York: Mc Graw HillLevinson, W. (2006). Review of Medical Microbiology and Immunology. San Francisco,California: Lange Medical Books/

McGraw-Hill Medical Publishing Division

http://en.wikipedia.org/wiki/Main_Page

http://www.textbookofbacteriology.net>search

http://www.cdc.gov/groupbstrep/guidelines/slidesets.html

http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm

http://microblog.me.uk/

http://student.ccbcmd.edu/~gkaiser/

http://www.spjc.edu/hec/VT/VTDE/ATE2639LGS/gramstain.htm

http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%204%20LB/Ch4Lessonbuilder10.htmlhttp://faculty.mc3.edu/jearl/ML/index.html

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/flora/Gram.html

http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/101lab6.html

http://lifesci.rutgers.edu/genmicrolab/index.htm

http://amrita.vlab.co.in/?sub=3&brch=76&sim=1109&cnt=1

http://web.med.unsw.edu.au/cdstest/etc.







http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%204%20LB/Ch4Lessonbuilder10.html

http://faculty.mc3.edu/jearl/ML/index.html





http://web.med.unsw.edu.au/cdstest/

http://web.med.unsw.edu.au/cdstest/





http://faculty.mc3.edu/jearl/ML/index.html

http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%204%20LB/Ch4Lessonbuilder10.html










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