CARA PEMBUATAN MEDIA

PADA MICROBIOLOGY

Selvianti P Djarudju
Specimen collection
isolation

5 “I” s
inoculation
inspection

identification
incubation
 Media
 A microbiologist can fine tune a media for almost
any purpose.
 General purpose media are designed to grow a
board spectrum of microorganisms e.g.
nutrient agar/broth, brain and heart infusion,
trypticase soy agar (TSA)-casein, soy digest and
NaCl.
 Enriched media has complex organic
substrates such as blood serum and special growth
factors (vitamins, amino acids).
 A bacteria that requires requires complex growth
factors is termed fastidious.
 Enrichment media facilitates the extensive
growth of particular organism.
 Base Media
 Breeding base medium simple breeding
that contains general substances that
need by large part microorganism and put
also as fundamental component to make
medium breeding other.
 Enrichment Media
 The addition of blood, serum extract or tryptic
soy agar will support the growth of many
fastidious microorganisms.
 Blood agar has the addition of citrated blood to
tryptic soy agar to make possible variable hemolysis
which permit the differentiation of some bacteria.
 α hemolysis: greenish brown halo around the colony ( e.g.
Streptococcus gordonii or S. pneumoniae)
 β hemolysis: complete lysis of blood cells resulting in a
clearing effect around the colony (e.g. Staphylococcus
aureus, Streptococcus pyogenes)
 Non hemolytic: no change in media (Staphylococcus
epidermidis, Staphylococcus saprophyticus
 Enriched Media
Chocolate agar, a
medium that gets brown
from heated blood. Used
for isolation of N.
gonorrhoeae.

Blood agar plate with

bacteria from human
throat. This media
differentiates among
different colonies by
appearance 6
 Selective/Differential Media
 A selective media contains agents that inhibit
the growth of certain microorganisms and select
for the growth of others.
 A selective media is important as a primary
isolation of specific organisms.
 E.g mannitol salt agar (MSA) has a high NaCl
concentration (7.5%) which will inhibit the growth
of most microbes but will select for the growth of
Staphylococcus.
 MacConkey agar which contains bile salts as a
selective agent.
 Selective/Differential Media
 Bile salts is a component of feces and inhibit the
growth of Gram positive bacteria and
encourage the growth of Gram negative rods.
 Other selective agents:
 Methylene blue and crystal violet inhibit Gram
positives
 Selenite and brilliant green are used in media
to isolate Salmonella from feces.
 Sodium azide is used to isolate enterococci from
water and food.
 The Conditions at Microbial Can
Grow Optimum In A Medium That
is:
 Medium must contain all easy nutrients be
used by microbial.
 Medium must has pressure osmosis,
surface and pH appropriate
 Medium doesn't contain hindrance
substance
 Medium must sterile
General vs Selective Media
Differential Media
 Differential media
grow several types of
organisms and
display visible
differences among
organisms.
 Differences may show
up as colony size,
media colour, gas
bubble formation and
precipitate formation.
Selective/Differential Media

MacConkey agar differentiates

Mannitol salt agar is between lactose-fermenting
used to isolate bacteria and (pink-red centre) and
members of the genus lactose-negative bacteria ( no
pink colouration).
Staphylococcus
♦ Triple sugar iron agar (TSIA). Differential
This media contain
Media
♦
fermentable carbohydrates
♦ Red phenol to indicate pH
change
♦ Iron that indicate H2S gas
production.
♦ Rxns (left to right) are:
♦ No growth
♦ Growth with no acid
♦ Acid production in the
bottom only
♦ Acid production in the
bottom and H2S gas
formation (black)
Chromagar orientation Differential
uses colour-formation to Media
distinguish at least 7
common urinary
pathogens.
This allow for rapid
identification and
treatment.
 In this example, the
bacteria were streaked
as to spell their names.
 Characteristic Media
 Characteristic media are used to test
bacteria for particular activity, product or
requirement.
 E.g. urea broth used to detect for
urease.
 Kinds of medium is Medium of Potato
Dextrosa Agar (PDA), PDB, NA, NB, LB,
LA, TEA, AND TEB.
.
Pembuatan Media
NA (Nutrien Agar) Untuk Bakteri

Peptone………….................................................. 5,00 g
Beef Extract ..........................................................3,00
Bacteriological Agar........................................... 15,00
Final pH 6,8 ± 0,2 at 25ºC

PDA (Potato Dextrose Agar) untuk Jamur

Potato(solids) ...................................................... 4,00 g

Dextrose............................................................. 20,00
Bacteriological Agar ...........................................15,00
Final pH 5,6 ± 0,2 at 25ºC
Direct Microscopic Examination
 Direct microscopic examination of a stained
specimen is often the most rapid method for
the identification of characteristics.
 Stains include Gram, acid fast, direct
fluorescent antibody test (DFA).
 DFA can be used to highlight the presences of
microorganisms in a specimen.
 DFA test are available for Staphylococcus aureus,
Streptococcus pyogenes, Neisseria gonorhoeae
and Haemophilus influenza.
 Direct Examination

Micrococcus luteus

E. coli (white), Micrococcus luteus

(yellow), Serratia marcescens (red)
Serratia marcescens
Direct Microscopic Examination
 Direct
Fluorescent
Antibody Test
 Biochemical Tests
 The microbe is cultured in a media with a special
substrate and tested for an end product.
 Prominent biochemical tests include carbohydrate
fermentation, acid or gas production and the
hydrolysis of gelatin or starch.
 Many of these test in rapid miniaturized system that
can detect for 23 characteristics in small cups called
Rapid test.
 The info from the rapid test are input into a
computer to help in identification of the organisms.
Carbohydrate  . This medium show
Fermentation fermentation (acid
production) and gas
formation.
 The small Durham tube
for collecting gas bubbles.
 Left- right:
 Uninoculated negative
control
 Centre, positive for
acid (yellow) and gas
(open space).
 Growth but no gas or
acid.
 Methyl Red Test
 This is a qualitative
test for acid
production.
 The bacteria is grown
in MR-VP broth.
 After addition of
several drops of
methyl red solution
a bright red colour is
positive and yellow-
orange negative.
Nitrate Reduction ♫ After 24-48 hrs of
incubation, nitrate
reagents are added.
♫ Left to right:
♫ Gas formation
(positive for nitrate
reduction).
♫ positive for nitrate
reduction to nitrite
( red colour).
♫ Negative control
 Starch Hydrolysis
 After incubation on
starch agar, plates
are flooded with
iodine solution.
 Positive test indicated
by colourless area
around growth.
 Negative test
indicated below.
 Catalase Test
 Place a drop of H2O2
on the culture.
 A positive reaction
show gas bubbles.
 Often used to
differentiate
Streptococcus
from
Staphylococcus.
 Biochemical Tests
 Other biochemical tests of interest include:
H2S production
Indole test
Oxidase test
Oxidation fermentation
Phenylalanine deaminase test
Antibiotic susceptibility tests
 Principle, procedure, most common use.
 Rapid Tests
 Rapid test: a biochemical system for the
identification of Enterobacteriacae and other
Gram –ve bacteria.
 It consist of plastic strips with 20 μl of
dehydrated biochemical substrates used to
detect biochemical characteristics.
 The biochemical substrates are inoculated with
pure cultures and suspended in physiological
saline.
 After 5 hrs-overnight the 20 tests are converted to
7-9 digital profile.
Rapid Test GLU--4

Results
OXI--0 +
- GEL-0 4
ARA-2 2 -
+ VP--0
AMY-0
-
IND--4
+
MEL--4 TDA-0 4
+ -
SAC-2 7 URE-0
+ -
RHA-1
+
H2S--0
-
SOR--4 CIT-0 1
+ -
INO-0 5 ODC-1
- +
MAN-1
+
LDC--4
+
normal 7 digit code ADH-0 5
-
5 144 572 = E. coli ONPG-1
+
 Bacteriophage Typing

 Bacteriophage typing is based on the specificity

of phage surface receptor for the cell surface
receptor.
 Only those phages that can attach to the
surface receptors can cause lysis.
 The procedure involves:
 A plate is heavily inoculated so that there is
no uninoculated areas.
 The plate is marked off in squares (15-20 mm)
and each square inoculated with a drop of
suspension for different phages.
Heavily Inoculated Plate
 Bacteriophage Typing
 The plate is incubated for 24 hrs then
observed for plaques.
 The phage type is reported as a specific
genus and species followed by the types
that can infect the bacterium.
 E.g. 10/16/24 means that the bacteria is
sensitive to phages 10, 16 and 24.
 Phage tying remain a tool for research and
reference labs.
 Unculturable Organisms

 Environmental researchers estimate

that < 1% of microorganisms are
culturable and therefore it is not
possible to use phenotypic methods of
identification.
 These microorganisms are called viable
nonculturable (VNC).
 Immunological Methods
 The study of antibody (Ab)- antigen (Ag) rxns in
vitro is called serology.
 Serological rxns are the basic of immunological
identification and diagnostic methods.
 The usefulness of serological test is dependent on
its sensitivity and specificity.
 Sensitivity is the ability to detect minute
amounts of Ab or Ag.
 Specificity is the ability to detect a single Ag or
Ab.
 False Negatives/Positives
 High sensitivity prevents false
negatives.
 False negatives occurs when there is
not reaction when the Ag or Ab is
present.
 High specificity prevents false positives.
 False positives occurs when there is
cross reaction with another molecule.
 Precipitation Reactions
 Precipitation (ppt) is the interaction of a soluble Ag
with an soluble Ab to for an insoluble complex.
 The complex formed is an aggregate of Ag and Ab.
 Ppt rxns occurs maximally only when the optimal
proportions of Ag and Ag are present.
 Ppt can also be done in agar referred to as
immunodiffusion.
 Ppt test uses antibodies to detect for streptococcal
group antigens.
Precipitation Reactions
 Agglutination Reactions
 Agglutination (Aggn) is the visible
clumping of an Ag when mixed with a
specific Ab.
 Aggn tests are widely used because they are
simple to perform, highly specific,
inexpensive and rapid.
 Standardized tests are available for the
determination of blood groups and
identification of pathogens and their
products.
Agglutination Reactions
 Genotypic methods
 Genotypic methods of microbe
identification include the use of :
Nucleic acid probes
PCR (RT-PCR, RAPD-PCR)
Nucleic acid sequence analysis
rRNA analysis
RFLP
Plasmid fingerprinting.
 Nucleic Acid Probes
 A single stranded probe is added and if there is
sequence complementality between the target
and the probe a positive hybridization signal will be
detected.
 Hybridization is detected by a reporter molecule
(radioactive, fluorescent, chemiluminescent) which
is attached to the probe.
 Nucleic acid probes have been marketed for the
identification of many pathogens such as N.
gonorrhoeae.
 Polymerase Chain Reaction (PCR)
 PCR is widely used for the identification of
microorganisms.
 Sequence specific primers are used with PCR in
the amplification of DNA or RNA of specific
pathogens.
 PCR allows for the detection even if only a few
cells are present and can also be used on viable
nonculturables (see sensitivity table).
 The presence of the appropriate amplified PCR
product confirms the presence of the organisms.
 Primers are available for the identification of Niesseria
gonorrhoeae, and to monitor food for the presence of
Salmonella and Staphylococcus.
 Sensitivity of Microbe Detection Tests

Methods Toxin or organism Sensitivity

Flow Cytometry S. Typhimurium in milk 103/ml in 40 min
Fluorescent Salmonella 106/ml
antibody
Latex agglutination E. coli enterotoxin 32 ng/ml

ELISA C. perfringens type A 1 ng/ml

toxin
PCR E. coli 1-5 cells/100 ml
of H2O
 Real Time PCR and RT-PCR
 Currently many PCR tests employ real time PCR.
 This involves the use of fluorescent primers.
 The PCR machine monitors the incorporation of the
primers and display an amplification plot which can be
viewed continuously thru the PCR cycle.
 Real time PCR yields immediate results.
 Another application of PCR is RT-PCR (reverse trancriptase
PCR).
 During RT-PCR an RNA template is used to generate
cDNA and from this dsDNA is generated.
 The enzyme used is reverse transciptase.
 RT-PCR is used to detect for HIV and to monitor the
progress of the disease.
RT-PCR
 RAPD-PCR
 Random amplified polymorphic DNA PCR uses a
random primer (10-mer) to generate a DNA
profile.
 What are random primers?
 The primer anneals to several places on the DNA
template and generate a DNA profile which is used
for microbe identification.
 RAPD has many advantages:
 Pure DNA is not needed
 Less labour intensive than RFLP.
 There is not need for prior DNA sequence data.
 RAPD has been used to fingerprint the outbreak of
Listeria monocytogenes from milk.
 PCR vs
RAPD-PCR
 DNA sequencing
 The determination of a small amount of DNA
sequence can be used for microbial identification.
 The most common sequence used for microbe
identification is DNA sequence of the 16S rRNA
gene.
 PCR is used to amplify the 16S rRNA gene and the
sequence determined.
 rRNA is a major component for ribosome and
ribosome have the same function in protein
synthesis in all cells.
 DNA Sequencing
 Computer analysis of 16S rRNA sequence has
revealed the presence of signature sequences,
short oligonucleotides unique to certain groups
of organisms and useful in their identification.
 rRNA sequence can be used to fine tune identity at
the species level e.g differentiating between
Mycobacterium and Legionella.
 16s rRNA sequence can also be used to identify
microorganisms from a microbial
community.
 Restriction Fragment Length
Polymorphism
 RFLP involves digestion of the genomic
DNA of the organism with restriction
enzymes.
 The restricted fragments are separated by
agarose gel electrophoresis.
 The DNA fragments are transferred to a
membrane and probed with probes
specific for the desired organisms.
 A DNA profile emerges which can be used
for microbe identification.
 RFLP of M.
tuberculosis
from 17
patients
 Plasmid fingerprinting
 What is a plasmid?

 Plasmid fingerprinting identifies microbial species

or similar strains as related strains often contain
the same number of plasmids with the same
molecular weight.
 Plasmid of many strains and species of E. coli,
Salmonella, Camylobacter and Psseudomonas has
demonstrated that this methods is more accurate
than phenotypic methods such as biotyping,
antibiotic resistance patterns , phage typing and
serotyping.
 Plasmid fingerprinting
 The procedure involves:
 The bacterial strains are
grown, the cells lysed and
harvested.
 The plasmids are
separated by agarose gel
electrophoresis
 The gels are stained with
EtBr and the plasmids
located and compared.