Microbiology Guide - Introduc FK-Unisba

Fakultas Kedokteran UNISBA Page 1

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 m to giant bacteria
larger than 700 m. 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 view, as
pathogenic 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 todays perspective this is a horrendous statistic, 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 science.

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 bodies
than 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 microbial 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.

http://www.microbiologytext.com/index.php?module=Book&func=displayarticle&art_id=643


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)
Enterococcus sp.
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.
Fusobacterium sp.

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.


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 does
not 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 dont allow to boil






Escherichia coli (Gram-negative rods)
Magnification: 1000
Klebsiella pneumoniae & Staphylococcus aureus
Magnification: 1000
Gram-negative rods and gram-positive cocci

(Gram-positive rods)
Magnification: 1000
Moraxella catarrhalis
Magnification: 1000
Gram-negative diplococci


Most isolation media are a combination of two or more types listed above.
Name of
Media
Type Use in Medical Lab Ingredients Growth Allowed Growth Inhibited
Trypticase
Soy
Agar/Broth
TSA, TSB
General
All Purpose
Basic 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
Mannitol
Salt 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

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. Lactosefermening 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 H
2
S 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 blueblack 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

Staphylococcus
aureus

Lowenstein-Jensen Media Agar
for Mycobacterium

Mycobacterium tuberculosis colonies
Different mycobacteria species grown on TB-Medium
Base according to Lwenstein- Jensen

Mueller-Hinton agar (MHA)
Use : For antimicrobial susceptibility testing of a variety of
nonfastidious, rapidly-growing microorganisms.

Spirit Blue agar

Use : This agar is used to identify organisms that are
capable of producing the enzyme lipase.

This enzyme is secreted and hydrolyzes
triglycerides to glycerol and three long chain fatty
acids. These compounds are small enough to pass
through the bacterial cell wall. Glycerol can be
converted into a glycolysis intermediate. The fatty
acids can be catabolized and their fragments can
eventually enter the Krebs cycle. Spirit blue agar
contains an emulsion of olive oil and spirit blue dye.
Bacteria that produce lipase will hydrolyze the olive
oil and produce a halo around the bacterial growth.
The Gram-positive rod, Bacillus subtilis is lipase
positive (pictured on the right) The plate pictured on
the left is lipase negative.

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


Hemolysis




Tests used to identify
Gram Positive Bacteria

Catalase Test
Mannitol Salt Agar (MSA)
Blood Agar Plates (BAP)
Motility Agar
Coagulase Test
Taxos 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)
Kligers Iron Agar (KIA)
Nitrate Broth
Motility Agar
MacConkey agar
Simmons Citrate Agar
Urease test
Sulfur Indole Motility Media (SIM)

Biochemical identification

The Controls That Were Performed
For The Various Tests


Urease test

Indole Test
ndole tests looks for the presence or
absence of tryptophanase enzyme
production of the bacteria. If the enzyme is
present,it will degrade the aminoacid
tryptophan in the media and will produce
Indole,ammonia and pyruvic acid. Indole will
react with Kovacs reagent to produce a
cherry red complex,which indicates a
positive indole test. The absence of red color
is indicative of tryptophan hydrolysis due to
the 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
produc
tion

Uninoculated
Tube

Negative
Urease
producti
on


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,G
Glucose 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,H
2
S Glucose fermentation only, Gas produced, H
2
S produced.
7 Yellow/Yellow with bubbles and black precipitate A/A,G,H
2
S
Glucose and lactose and/or sucrose fermentation, Gas
produced, H
2
S produced.
8 Red/Yellow with black precipitate K/A,H
2
S Glucose fermentation only, H
2
S produced.
9 Yellow/Yellow with black precipitate A/A,H
2
S
Glucose and lactose and/or sucrose fermentation, H
2
S
produced.


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 such
an 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 energy
source. 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 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

It is broken down
by gelatinase into smaller
polypeptides, 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 pyruvic
acid 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

Susceptibility test

For this example the MIC for erythromycin is 0.19 g/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 g/ml = susceptible, 0.5
g/ml = intermediate, and 1.0 g/ml = resistant; and for Erythromycin: 0.25 g/ml = susceptible, 0.5 g/ml = intermediate, and 1.0 g/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, 1618 mm = intermediate, and 15 mm = resistant; for erythromycin: 21 mm = susceptible,
1620 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.

Depicted above are two methods for
determining antimicrobial susceptibility of
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 is
called 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 growth
meets the strip.

Taxos A (bacitracin sensitivity testing)

This is a differential test used to distinguish between organisms
sensitive to the antibiotic bacitracin and those not. Bacitracin is a
peptide 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 streaked
with Streptococcus pyogenes; notice the large zone of inhibition
surrounding 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).

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.

Carbohydrate
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

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)


Protein Catabolism (Proteolysis)

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
Barrits reagent A ( 40% KOH) and
Barrits 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 Barrits 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 test

This is a differential medium. It is used to determine if an
organism is capable of reducing nitrate (NO
3
-
) to nitrite
(NO
2
-
) or other nitrogenous compounds via the action of the
enzyme nitratase (also called nitrate reductase). This test is
important in the identification of both Gram-positive and
Gram-negative species.

After incubation, these tubes are first inspected for the
presence of gas in the Durham tube. In the case of
nonfermenters, this is indicative of reduction of nitrate to
nitrogen gas. However, in many cases gas is produced by
fermentation and further testing is necessary to determine if
reduction of nitrate has occurred. This further testing
includes the addition of sulfanilic acid (often called nitrate I)
and dimethyl-alpha-napthalamine (nitrate II). If nitrite is
present in the media, then it will react with nitrate I and
nitrate II to form a red compound. This is considered a
positive result. If no red color forms upon addition of nitrate I
and II, this indicates that either the NO
3
-
has not been
converted to NO
2
-
(a negative result), or that NO
3
-
was
converted to NO
2
-
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 any
remaining NO
3
-
to NO
2
-
thus allowing nitrate I and nitrate II
to react with the NO
2
-
and form the red pigment (a verified
negative result). If no color change occurs upon addition of
zinc then this means that the NO
3
-
was converted to NO
2
-
and then was converted to some other undetectable form
of 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 pictured
on the left) after the addition of Zn, this indicates a negative
result. If there is no color change in the tube after the
addition of nitrate I and nitrate II, the result is uncertain. If
the tube is colorless (picture on the right) after the addition
of Zn this indicates a positive test.


Bile esculin test

Bile Esculin Agar is recommended
for the presumptive isolation and
identification of group D
streptococci.

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 -hemolytic
streptococci by demonstrating its
susceptibility 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 area
become 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
hydrolyze starch (amylose and amylopectin) using
the enzymes -amylase and oligo-1,6-glucosidase.
Often used to differentiate species from the
genera Clostridium and Bacillus. Because of the
large size of amylose and amylopectin molecules,
these organisms can not pass through the bacterial
cell wall. In order to use these starches as a carbon
source, bacteria must secrete-amylase and oligo-
1,6-glucosidase into the extracellular space. These
enzymes break the starch molecules into smaller
glucose subunits which can then enter directly into
the glycolytic pathway. In order to interpret the
results of the starch hydrolysis test, iodine must be
added to the agar. The iodine reacts with the starch
to form a dark brown color. Thus, hydrolysis of the
starch will create a clear zone around the bacterial
growth.Bacillus subtilis is positive for starch
hydrolysis (pictured on the left). The organism shown
on the right is negative for starch hydrolysis.
Starch hydrolysis test


Litmus milk
1 control, 2 pink = acid, 3 white = reduction, 4 & 5 stormy fermentation, 6 blue = alkaline
Litmus milk test
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 various
changes. 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 (bound
to DNA)
+ = colorless zone around organism
Lipase spirit blue agar
lipid
(triglyceride)
glycerol, fatty acids

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

Selective and Differential Media
Test Medium Substrate
Selective
Ingredient
pH
indicator
Result + organisms
EMB agar selective for
gm - & differential for
lactose fermentation
Eosin
Methylene
Blue (EMB)
agar
Lactose
Eosin +
Methylene Blue

Growth = gm -;
Dye accumulation (color) in organism
= lactose fermentation:
Metallic green
Fish eyes (pink with purple center)
E. coli
E. aerogenes
MacConkeys agar
selective for gm - &
differential for lactose
fermentation
MacConkeys
agar
Lactose
Crystal violet +
bile salts
Neutral red
in agar
Growth = gm -;
Pink ppt. in organism = lactose fermentation
E. coli;
E. aerogenes
Mannitol Salt Agar
(MSA) selective for staph
& differential for mannitol
fermentation
MSA Mannitol 7.5% NaCl
Phenol red
in 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 Kovacs layered on top Red at surface E. coli
Methyl 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 (Barritts) Red throughout E. aerogenes
Citrate Utilization Simmons citrate agar Citrate Na
2
CO
3
Bromthymol
blue in agar
Royal blue E. aerogenes

TSI (Triple sugar iron)
Test Media Substrate Product(s) Reagent pH indicator + appearance + organisms
H
2
S Gas TSI agar
(stab & drag)
Cysteine H
2
S Iron in media Black ppt. when H
2
S reacts with iron
to form iron sulfide
S. typhimurium
P. vulgaris
Carbohydrate
Fermentation
TSI agar
(stab & drag)
Glucose, lactose,
sucrose
Organic acids Phenol red in
agar
Media: yellow = acidic (A);
red = alkaline (K);
Record reactions as slant/butt:

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

Carbohydrate Fermentation Series
Test Media Substrate Product(s) pH indicator + appearance
Carbohydrate Fermentation Carbohydrate broth
with Durham tubes
Glucose, lactose, or sucrose Organic acids Phenol red in broth Acidic (yellow) media
Gas Production CO
2
Bubble in Durham tube

Test Media Substrate Product(s) pH indicator + appearance + organisms
Urease (Christensens) urea agar Urea Ammonia, CO
2
Phenol red in agar pink (alkaline) media Proteus
Litmus Litmus milk Casein, lactose Various Litmus in media pink = acid; sugar fermentation
hard curd = acid
soft curd = rennin clots casein
blue = alkaline; casein breakdown
brown = further casein breakdown
white = litmus reduction


Test Substrate Product(s) Reagent + appearance + organisms
Catalase Hydrogen peroxide
(H
2
O
2
)
Water, oxygen Bubbles Staphylococci
Coagulase Fibrinogen Clumping factor-fibrinogen complex Staphyloside latex beads Blue ppt. (clumping) S. aureus
(Cytochrome)
Oxidase
Cytochrome c Oxidized cytochrome c Phenylenediamine Blue color P. aeruginosa
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 zone
around/beneath organisms
Strep. pneumoniae
E.(S) faecalis
Staph. epidermidis
**Beta
Blood agar (5% SB) RBCs & hemoglobin (Hb) Hemolysis; complete digestion of Hb Clear zone around organisms Strep. pyogenes,
E.(S) faecium
Staph. aureus
Gamma Blood agar (5% SB) RBCs & hemoglobin (Hb) No hemolysis No clearing around
organisms

*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;
N
2
/ammonia
Nitrate A & B, zinc
added 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 and nitrite reductase
If red after zinc = neither nitrate or nitrite reductase

Nitrate reductase Nitrite reductase
Nitrate (NO
3
) Nitrite (NO
2
) N
2
(gas) /NH
3
(ammonia)

Nitrate A & B
+ nitrate = colorless
Zinc
reduces nitrate to nitrite

Nitrate A & B
+ nitrite = red

Nitrate A & B
+ N
2
(gas) or NH
3
(ammonia) = colorless



Bacterial Infection (Secondary Infection)
(sign / symptoms)
(Etiologic agent)
(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 growth
of Gram-negative, small rod-shaped and facultatively anaerobic
String test of a Klebsiella pneumoniae isolate
demonstrating hypermucoviscosity

Pneumonia caused by Klebsiella pneumoniae.
K. pneumoniae can cause the disease Klebsiella pneumonia. They cause
destructive changes to human lungs inflammation and hemorrhage with cell death
(necrosis) that sometimes produces a thick, bloody, mucoid sputum (currant jelly
sputum). 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 Negative Bacillus
Cut surface of normal lung

Tripple Sugar Iron Medium (TSI medium)
Yellow/Yellow (acidic)
with bubbles
A/A,G
Glucose and lactose and/or
sucrose fermentation, Gas
produced.

Klebsiella pneumoniae
biochemical test :
Indole positive
Urease positive
Citrate positive
Voges-Proskaueur positive
TSI : yellow/yellow, gas (A/A,G)

uninoculated A/A,G
Positive
citrate
Negative
citrate
Urease
test
Indole test

References :

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco: Pearson Education
Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in general microbiology. (10th ed.). New
York: Mc Graw Hill
Levinson, 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.html
http://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.

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