MICROBIOLOGY STUDY TIPS

CSMLS COMPETENCIES RELATED TO MICROBIOLOGY

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To get ready for Microbiology Competencies, I would suggest focusing on the following topics in
addition to reading/preparing to obtain a strong knowledge base.

I. MICROBIOLOGY OVERVIEW:
The specimen processing in Microbiology includes processing specimens from various body
sites for the isolation and identification of pathogens, if present. Important points to note:
a. Sources/body sites of specimens for microbiological specimens, and the knowledge of
each of these specimens are collected and processed; expected pathogens from each site.
These sites include, but are not limited to:
i) Blood
ii) Respiratory specimens: Throat swab, Sputum, Bronchioalveolar lavage (BAL),
Bronchial wash, Transtracheal aspirate (TTA), Lung biopsy
iii) Nervous system specimens:
iv) Urinary tract specimens: Mid-stream urine, Catheter specimen, Pediatric
specimen, Suprapubic aspiration (directly from bladder)
v) Gastrointestinal tract (GIT, Enteric) specimens: Stool, rectal swab
vi) Genital tract specimens: Female: Vaginal swab, cervical swab; Male: penile swab,
semen, prostatic secretion
vii) Skeletal muscle/bone/joint: Aspirate or biopsy
viii) Wound (Superficial, subcutaneous or deep seated): Swab or Aspirate
ix) Other sites of infection such as pleural, peritoneal, joint fluids, ear swab, eye
swab, tissues, etc
b. Differentiation in sterile and non-sterile body sites/sources
c. Names and identification characteristics of probable pathogens that can cause infection
at each site. Note: remember that a pathogen at one site can be normal flora at another.
For example, Proteus species are a prominent group of pathogens in urinary tract, but are
a part of the normal flora in gastrointestinal tract.
d. Methods of processing appropriate for each specimen: Gram stain or any other special
stain; media selection, inoculation and incubation, examination of isolates to identify
pathogens if present, susceptibility tests, susceptibility testing, interpretation and
reporting.
e. Antimicrobial Susceptibility Tests

II. SPECIMEN COLLECTION AND TRANSPORT

f. Specific protocols for collection and transport of specimens from different body sites. It is
important to know the criteria for rejection of specimens which do not follow the
collection and transport protocols such as the timeline, using an appropriate transport
medium, aseptic collection technique..
g. Receiving a specimen in logbook/LIS – request a new specimen for those rejected

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III. SPECIMEN PROCESSING: DAY 1

h. Gram Stain: Principle and troubleshooting of Gram stain is of utmost significance. Gram
stain morphology of the specimen is the most significant first step in the identification and
isolation of pathogens, as well as for the assessment of the quality or acceptability of the
specimen. Interpretation of Gram stain is a critical step, especially on sterile specimens
such as CSF or blood culture. It should be noted that Gram stain is not indicated on some
specimens such as throat swab and urine specimens unless specifically indicated by the
physician. On the other hand, Gram staining and wet preparation of vaginal swab is an
example of how an interpretation of Gram stain can lead to diagnosis of abnormal
conditions. Gram staining of sputum is also used for grading the specimen and deciding its
acceptability.

Acid-fast stain or any other special stain such as immunofluorescence tests or staining of
fecal specimens for the detection of parasites, if specifically requested – for example,
modified acid-fast stain for Cryptosporidium parvum in feces.

i. Culture media routinely used for specimens from various sites. The selection of media
depends on the likely pathogens that can cause infection at that site. Therefore, a set of
media is selected in such a way that any of the likely pathogens can be isolated on at least
one, or more media. Culture media for specific or fastidious pathogens not routinely
encountered are used only when requested by the physician, or when other findings
indicate the presence of a particular pathogen.
j. Types and composition of media: Blood agar (most commonly sheep), chocolate agar,
and MacConkey agar are the three most commonly used media for isolation of pathogens
when aerobic/facultative Gram positive cocci and bacilli, Gram negative diplococci, and
Gram negative bacilli and coccobacilli are expected from clinical specimen(s). Special
media need to be used when fastidious organisms which are difficult to be isolated from
non-sterile specimens. For example, Corynebacterium diphtheriae, Bordetella pertussis,
Legionella spp, anaerobes. The knowledge of purpose and components of these media is
essential to justify their use.
Some special/selective media are used for the isolation of pathogens from specimens such
as urine and feces. Principles of their use, components, selective agents if present,
differential agents (e.g. carbohydrate-indicator combinations) must be known to be able
to differentiate between pathogens and normal flora/contaminants.
Transport media are used for the transporting specimens, chromogenic media are used
for screening or identifying specific bacteria.
Another group of media includes those used for biochemical identification tests and
screening tests in case of enteric pathogens.
Blood culture media and automation is a separate category that one should be aware of.
k. Inoculation and incubation of culture media: It involves the knowledge of environmental
conditions required for each of the expected pathogen – strict aerobes, facultative
anaerobes, microaerophilic organisms, and strict anaerobes. Optimum incubation
temperatures vary for some bacteria from the most common 350C, and need to be

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 addressed accordingly. The length of incubation can vary. Although most routinely
isolated human pathogens produce visible colonies within 24 hours of incubation, some –
such as Neisseria gonorrhoeae or Burkholderia cepacia may need longer, as well as
actinomycetes such as Nocardia sp.
Mycobacteria have totally different requirements for isolation and identification.
Some specimens such as urine need a quantitative culture method to differentiate
between infection and contamination.
l. Non-cultural methods for expected pathogens that do not grow on culture media:
Special microscopic techniques such as Dark Field, Fluorescent staining; Immunological
methods of detection of antigens and/or antibodies;, Molecular/Nucleic acid detection
techniques; or other advanced techniques such as MALDI-TOF. Examples: Chlamydia,
spirochetes such as Treponema pallidum, other infections that need shorter turn-around-
time.

IV. SPECIMEN PROCESSING: DAY 2

m. Identification of isolated pathogens/differentiation from normal flora or contaminants:

Traditional identification tests on the isolated include:

i) Gram stain is the first step towards identification of the isolate by grouping them into
specific groups such as Gram positive cocci – with their arrangements as in clusters,
chains, pairs; Gram positive bacilli – long/short, in chains, palisades, with/without spores;
Gram negative diplococci; Gram negative coccobacilli, Gram negative bacilli , and other
morphologies such as curved or spiral bacteria, anaerobes, yeast and other clinically
significant fungi such as Aspergillus spp, Pneumocystis jiroveci
ii) Examination of colonial morphology on different media and their correlation to Gram
stain
iii) Biochemical tests: These are varied, and many depend on the results of Gram stain
and/or colony morphology. Knowledge of which tests are performed for which group of
organisms; as well as principles/media/interpretation of each is essential.
Correlation with Gram stain, colony morphology and Identification methods/charts for
common groups of aerobic and facultative bacteria such as Gram positive cocci in (GPC)
clusters/pairs/chains; Gram negative cocci in pairs (GNDC); Gram positive bacilli (GPB),
Gram negative bacilli (GNB); and other bacteria with specific morphologies; anaerobes,
yeasts and other fungi
iv) Serological tests using specific antisera for the detection of specific bacterial antigens e.g.
Streptococcal grouping, enteric pathogens such as Salmonella spp, Shigella spp,
pathogenic E. coli
v) Special tests for the confirmation of a pathogen: Tests for detection of toxins by
Corynebacterium diphtheriae, Clostridium difficile, Clostridium perfringens

Newer technologies for identification: Automated methods such as VITEK ; Molecular

techniques such as PCR (Polymerase Chain Reaction) and related techniques, and MALDI-TOF
(Matrix Assisted Laser Desorption/Ionization – Time of Flight) are examples of newer
technologies that are used for the identification of pathogens, especially those that do not

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 grow easily on culture media, or take a very long time to grow. These faster techniques
improve patient outcomes due to shorter turn-around-times. The knowledge and
troubleshooting these techniques are expected part of competence of an MLT.

vi) Interpretation of isolates

In case of sterile specimens, any isolate is considered significant after ruling out
contamination of incorrectly collected specimens. For specimens that normally contain
normal flora, such as a sputum specimen, it is extremely important to differentiate
between normal flora and pathogens following specific criteria. To interpret these tests
correctly, the principles of and media used for the biochemical tests should be clearly
understood. Automated instruments such as VITEK are used for identification of
pathogens and for susceptibility testing.
vii) Susceptibility Tests: should be set up on day two for presumptively identified isolates.
viii) Critical results: Recognize and report critical results as soon as possible to the treating
physician. Example: isolation of a pathogen from a sterile site such as blood or CSF;
isolation of a pathogen that requires a higher level of safety precautions e.g. probable
Brucella spp, fungus such as Coccidioides sp.

IV. SPECIMEN PROCESSING: DAY 3

n. Interpretation and Reporting: The purpose of reporting is to help the healthcare team in
diagnosis. Therefore, the isolate should be reported only if it is the likely causative agent,
and not a normal flora. Reporting timelines and guidelines must be followed. If the report
is not ready for any reason on a due date, the physician must be sent a preliminary report.
Appropriate follow up about reporting to the Public Health Office of a pathogen listed
under Notifiable Diseases must be performed.
After the isolate is identified, antimicrobial susceptibility testing should be performed
according to guidelines.
o. Report susceptibilities: Read, interpret, and report susceptibility test results. Some may
need further follow up – for example, if MRSA is identified, it should be tested for
vancomycin MIC, probable ESBL should be confirmed by phenotyping.

V. Antimicrobial Susceptibility Testing (AST):

a. Antibiotics: Classes of antibiotics, their modes of actions, resistance mechanisms of
microorganisms. Examples: Beta lactam group, beta lactamase inhibitors, aminoglycosides,
macrolides….
b. Antibiotic panels: which antibiotics should be used as a first line of defense against a specific
pathogen/group of pathogens.
c. Criteria for performing susceptibility tests: For which isolates the AST must be performed, and
which have a predictable susceptibility against a particular antibiotic, and routine AST is not
required.
d. Methods of performing ASTs to detect Susceptibility or Resistance of an organism towards an
antimicrobial agent: Kirby-Bauer Disk Diffusion method (Qualitative to report S I R to a
concentration achievable at a body site); E-test strip method (Quantitative -gives MIC – minimum

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 inhibitory concentration of antibiotic needed to inhibit the causative agent), Tube/Plate dilution
methods (Quantitative).
e. Special methods for detecting resistance of a bacterial species or a group of bacteria e.g.
resistance to beta lactam antibiotics by some bacteria, specialized mechanisms such as MRSA,
VRSA, VRE, ESBL, CRE. Specific techniques for Gram Positive cocci such as penicillin resistance by
Streptococcus pneumoniae, or induced resistance to clindamycin in presence of erythromycin – D
zone test.
f. Principles, Techniques, Interpretation: Know the principles of detection, techniques, and
interpretation of all the above methods of detection of resistance.
g. Infection Control Measures: Screening of new patients/admissions for detecting carriers of
antibiotic resistant organisms (AROs); screening isolates for probable resistance patterns;
informing Infection Control Officer if any of the above is detected.
h. Quality control for each of the above methods.
i. Troubleshooting to identify the reasons for unacceptable results of Quality Control/patient
results.
VI. IMMUNODIAGNOSTIC TECHNIQUES:
Immunoassays and Immunodiagnostic techniques use the antigen-antibody reactions for the
detection of specific antigens and antibodies in produced as a cause of or in response to an
infectious disease. Various techniques are used – you can see these in 3 links provided in the
list.
VII. MOLECULAR BIOLOGY & TECHNIQUES
Overview: Molecular biology is the branch of biology that concerns the molecular basis
of biological activity in and between cells, including molecular synthesis, modification,
mechanisms and interactions.[1][2] The central dogma of molecular biology describes the
process in which DNA is transcribed into RNA then translated into protein, namely, the
processes of replication, transcription, translation, and cell function.
By integration into other disciplines such as Biochemistry and Genetics, molecular techniques
have a myriad of clinical applications. In microbiology, identification of bacteria by
amplification of a DNA sequence specific for a microorganism – for example, Polymerase
Chain Reaction (PCR).

What you need to study:

a. Structure of DNA & RNA, the processes of DNA replication in detail; and Transcription into RNA,
and translation to form proteins in brief.
b. Tools used in Molecular Biology: Gel electrophoresis, hybridization techniques such as
Southern/Northern/Western Blotting, DNA amplification techniques such as PCR, use of molecular
markers such as RFLP.
c. Polymerase Chain Reaction: Complete technique in detail with reagents, instrument used,
visualization methods (gel electrophoresis/hybridization techniques), interpretation of results,
Quality Control, Troubleshooting
d. Types of PCR, their principles and clinical applications: RT-PCR (Reverse Transcriptase PCR), Real
Time PCR (QPCR), Nested PCR, Multiplex PCR, Probe Amplification Techniques.

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VIII. MALDI-TOF

Matrix-assisted Laser Desorption/Ionization – Time of Flight (MALDI-TOF) is one of the newest

technologies adopted all over the globe for rapid and accurate identification of microorganisms
based on automated analysis of the mass distribution of microbial proteins.

What you need to know: Working principle of MALDI-TOF, brief technique, types of
microorganisms that can be identified by this method, limitations, advantages and disadvantages
of this technology, principle of Electrospray-Ionization-MS (ESI-MS).

IX. MICROSCOPIES: (Should be included in Histotechnology & Hematology as well)

a. Bright Field Microscopy: Detailed information about the parts of the microscope and function of
each. Understanding of Magnification, Resolution, Numerical Aperture (NA), Lenses and their
magnification, working distance, Setting up Koehler illumination and its purpose.
b. Other Microscopies: As listed in the competencies - Their principles and applications.