Transcript Specimen Cultivation - Cal State LA

Specimen Cultivation
How do you grow these bugs?
Introductory remarks
 Although the future trends in clinical
microbiology are clearly pointing in the direction
of developing rapid, non growth dependent
means of detecting the presence of infectious
microorganisms (detection of microbial antigens,
probing for genes, PCR), the isolation and
identification of viable pathogens is still the
major means by which infectious diseases are
diagnosed today. This is particularly true for
bacterial infections.
Introductory remarks
 In order to identify an infectious bacterial
agent by biochemical methods and to do
antimicrobic sensitivity testing, it is
necessary to have a pure culture of the
organism. Why?
 The way that we do this in the clinical lab is to
streak a portion of the original sample out on
primary isolation media to get isolated
colonies.
Streaking for isolation
Introductory remarks
 Each isolated colony is a pure culture since it is
theoretically the progeny of a single cell.
 Each type of bacteria forms a characteristic colony
in terms of shape, size, color, texture, and
adherence to the medium.
 These colonial characteristics are often used in the
clinical lab as a first step in the process of
identification of bacteria.
Different bacterial colonial
morphologies
Different bacterial colonial
morphologies
Introductory remarks
 When the term culture media is used, it is
important to keep in mind that it refers to
artificial media on which bacteria and fungi
are grown.
 It is also important to remember that some
bacteria have never been successfully grown
on artificial lab media. For example:
 Treponema pallidum – is usually grown in the
testes of rabbits
Introductory remarks
 Mycobacterium leprae – is usually grown in the nine-banded
armadillo or in the foot-pads of mice.
 Some bacteria (Rickettsia and Chlamydia) and all viruses
are obligate intracellular parasites and can only be
grown in living host cells, such as whole organisms or
tissue culture cells.
 For viruses grown in tissue culture, identification is often based
on electron microscopic visualization of virus in the cells, or
 Cytopathic effects (visible damage to or changes in the cells)
produced by growth of the virus in the cells or by
 Inclusion bodies
 Syncytia formation
 Rounding up of the cells
Cytopathic effects – inclusion bodies
Cytopathic effects – syncytia formation
Cytopathic effects – cells rounding up
Introductory remarks
 Growth of obligate intracellular parasites is very time
consuming and expensive
 Diagnosis of these organisms as the causative agent
of an infection is usually by immunological means.
 In a direct assay one looks for antigens from the organism.
Therefore, one is looking for the organism, itself, usually in
tissues.
 In an indirect assay, one looks in the host for evidence of an
immunological response against the organism, usually in the
form of specific antibodies against the organism.
Introductory remarks
 When trying to diagnose an infection by assaying for antibodies
against the causative agent, it is best to take both an acute
(when patient is the most ill) and a convalescent (when patient
is recovering) phase specimen. Typically one looks for a 4-fold
rise in antibody titer (amount of antibody/ml of sera) between
acute and convalescent specimens. Why?
 Parasites are not easily grown on artificial media
or in living hosts.
 The diagnosis of parasitic infections is usually
based on the visual identification of the parasite
itself or on microscopic identification of the egg,
ova, or cyst produced by the parasite.
Taenia solium scolex
Taenia solium proglottids
Entamoeba histolytica trophozoite
Entamoeba coli cyst
Enterobius vermicularis egg
Ascaris lumbricoides egg
Plating of specimens and incubation of
cultures for bacteria and fungi
 When should the specimen be plated?
 The specimen should be plated immediately
because if there is a delay this may result in:
 A loss of fastidious or anaerobic organisms or
 There may be an overgrowth by normal flora in the
specimen resulting in a change not only in the
total number of microorganisms, but also in the
relative numbers of microorganisms. Why is this a
problem?
Plating of specimens and incubation of
cultures for bacteria and fungi
 What should the specimen be plated on?
 Diagnostic labs vary in their choices of routine plating
media that is used for growing bacteria and fungi
typically isolated from different types of specimens.
 The choices used by different labs take into account
what pathogens are anticipated in a given type of
specimen, their growth requirements ( CO2,
different temperature requirements, etc.) and the
peculiarities of the available media.
Example
Example
Plating of specimens and incubation of
cultures for bacteria and fungi
 Types of media that may be used include:
 General purpose media – this is media that
supports the growth of most common pathogens
and is therefore, classified as non-selective.
However, in plate form, this type of media permits
the isolation and differentiation of a wide variety of
bacteria via differences in colony size, shape, color,
texture, and adherence to the culture media.
Examples of general purpose media include:
Plating of specimens and incubation of
cultures for bacteria and fungi
 Nutrient agar
Plating of specimens and incubation of
cultures for bacteria and fungi
 Blood agar –this media also allows differentiation based
on the production by bacteria of hemolysins that destroy
red blood cells in the agar.
 Alpha () hemolysis is incomplete hemolysis and
appears as a green halo surrounding the bacterial
colony
 Beta () hemolysis is complete hemolysis and
appears as a clear area surrounding the bacterial
colony
 Gamma () hemolysis is no hemolysis at all
Types of hemolysis
Alpha
Beta
Gamma
Plating of specimens and incubation of
cultures for bacteria and fungi
 Chocolate agar – is essentially the same as blood agar,
except that the RBCs are lysed when added to the agar.
This releases hemin and NAD for utilization by fastidious
bacteria. The lysis gives the medium its chocolate brown
color.
Plating of specimens and incubation of
cultures for bacteria and fungi
 Some books call blood agar and chocolate agar “enriched
media” because the blood that is added to the media
acts to “enrich” the media.
 Selective media – this type of media contains
special nutrients that support the growth of certain
pathogens and/or inhibitors that suppress the
growth of competing normal flora. For example:
 Phenyl ethyl alcohol agar (PEA) – contains phenyl ethyl
alcohol to inhibit the growth of Gram negative bacteria
(Why does it inhibit the growth of G – bacteria?)
PEA agar
Plating of specimens and incubation of
cultures for bacteria and fungi
 Selective media is also usually differential. What makes it
differential is often the addition of a carbohydrate and a pH
indicator that together allow one to differentiate between
organisms that ferment the carbohydrate and those that do
not. For example:
 Mannitol salts agar (MSA)- this media contains:
 7.5% NaCl to suppress the growth of organisms that are
not halophilic (selection)
 Mannitol and the pH indicator phenol (for differentiation).
 If an organism that grows on the media ferments
mannitol, acid is produced and this lowers the pH. At a
low pH phenol red turns yellow. Therefore, organisms that
ferment mannitol turn the media yellow:
MSA
Plating of specimens and incubation of
cultures for bacteria and fungi
 MacConkey agar (Mac) – this media contains:
 Crystal violet to inhibit the growth of G+ bacteria and
fungi (selection).
 Lactose and the pH indicator neutral red which is red or
pink at an acid pH (differentiation).
 Organisms that are able to grow on the media and that
ferment lactose produce pink colonies on the media
 Organisms that don’t ferment lactose produce colorless
colonies.
 Mac plates are an example of what are collectively called
enteric agar plates because they facilitate the isolation and
differentiation of enteric pathogens. You will be discussing
other types of enteric agar plates in the laboratory.
Mac plate
Mac plate
Plating of specimens and incubation of
cultures for bacteria and fungi
 Reducing media – this type of media is used for
cultivating anaerobes. It contains compounds that
chemically combine with dissolved oxygen in the
media to deplete the O2 from the media. For
example:
 Sodium thioglycollate broth contains:
 Thioglycolic acid as a reducing agent to create an
anaerobic atmosphere deeper in the tube and
 Resazurin as an oxygen-reduction indicator. In the
presence of O2, resazurin turns pink. Where do
different type of organisms grow in the thioglycollate
broth?
Growth of different types of organisms in
thioglycollate broth
Plating of specimens and incubation of
cultures for bacteria and fungi
 Enrichment culture media – is used to prevent
missing a type of bacteria present in only small
numbers.
 This media is usually liquid and it provides nutrients and
environmental conditions that favor the growth of one
type of organism while being unsuitable for the growth
of others. For example, to enrich a stool culture for
enteric pathogens that may be found in low numbers
relative to the normal flora from the intestine:
 Gram negative broth
 Tetrathionate broth
 Selenite broth
Plating of specimens and incubation of
cultures for bacteria and fungi
 Special plating procedures
 Anaerobic cultures
 Reducing media may be used, or
 Plates can be incubated in a special jar or pouch in
an oxygen free atmosphere. In an anaerobic jar
the oxygen free atmosphere is generated by the
following mechanism:
Plating of specimens and incubation of
cultures for bacteria and fungi
 Envelopes containing sodium bicarbonate and borohydride
are placed in the jar and water is added and the chemical
reaction that occurs generates CO2 and H2.
 The H2 that is generated combines with O2 in the jar in the
presence of a palladium coated alumina pellet that acts as a
catalyst for the following reaction:
2H2 + O22H2O
Thus O2 is removed from the atmosphere.
 An indicator strip with methylene blue that is colorless in the
absence of oxygen and blue in the presence of oxygen is also
placed in the jar.
An anaerobic jar
Plating of specimens and incubation of
cultures for bacteria and fungi
 Blood cultures – as discussed previously, blood
cultures are usually inoculated into blood culture
media directly at the bedside of the patient.
 Frequently two bottles of liquid media are inoculated, one for
aerobic growth (TSB – tryptic soy broth) and one for
anaerobic growth (Thio).
 The bottles are usually examined every day for turbidity for
7-14 days.
 If turbidity develops, some of the media is removed for Gram
staining and subculturing onto solid media.
 Depending upon the lab policies, blood cultures may
routinely be Gram stained and subcultured at specific
intervals (24 hrs.,48 hrs, etc.) even in the absence of
turbidity.
Plating of specimens and incubation of
cultures for bacteria and fungi
 Some labs have machines (Bactec) that automatically detect
growth in blood cultures by  CO2 production
 Quantitative counts – as discussed previously, this is
often done on urine specimens.
 A known volume of specimen is plated on the agar medium,
via the use of a calibrated loop, and the number of colonies
that grow are counted.
 Caution – this only represents the number of bacteria
present at the moment of plating.
 For a clean catch urine specimen >100,000 colonies /ml is
considered significant and indicative of disease.
 For a bladder or kidney specimen >10,000 colonies/ml is
considered significant and indicative of disease. Why?
Quantitative urine counts
Plating of specimens and incubation of
cultures for bacteria and fungi
 Plating for unusual bacteria –
 Some of the more rarely encountered pathogens need
unusual media and/or techniques to facilitate their isolation.
 If a physician suspects that one of these organisms is the
causative agent of an infection, he/she must notify the lab so
that appropriate media can be prepared and proper
precautions can be taken, if necessary. For example:
 Brucella species (sp.)
 Bordetella sp.
 Legionella sp.
Plating of specimens and incubation of
cultures for bacteria and fungi
 Incubation of cultures
 Temperature
 Media inoculated with human specimens is usually incubated
at 35-370 C, which is the optimum growth temperature for
most human pathogens.
 Fungi are often grown at room temperature (RT).
 Many fungi are dimorphic and will grow as yeast at 370 C and
as molds at RT. Candida albicans is different in that at RT it
grows as a yeast, but at 370 C and in the presence of serum, it
grows as a mold.
Plating of specimens and incubation of
cultures for bacteria and fungi
 Atmosphere
 Most pathogenic bacteria grow best at 2-10% CO2
and most clinical labs routinely use 5% CO2
incubators for their cultures.
 Certain bacteria actually require 5-10% CO2 in
order to grow or to grow well (Neisseria sp.,
Streptococcus sp., Haemophilus sp.).
 To facilitate the growth of theses organisms, we will
grow them in candle jars to provide the higher CO2 that
they need for growth.
Candle jar
Plating of specimens and incubation of
cultures for bacteria and fungi
 Anaerobes have already been discussed
 Length of incubation time
 Most routine cultures are kept for 16-18 hours
(overnight) before being reported out as negative
 CSF and blood cultures are usually kept 1 week
before being reported out as negative
 Wound cultures may be kept for 48 hours before
being reported out as negative
Plating of specimens and incubation of
cultures for bacteria and fungi
 Fungal cultures are kept for 3-4 weeks before
reporting out as negative
 Cultures for Mycobacteria are kept for 6-10
weeks before reporting out as negative.