ABOUT MCS TESTS

1. MICROBIOLOGICAL CULTURE
SENSITIVITY TESTS
Microbiology:
• Microbiology is the study of living organisms that are invisible to the
naked eye , such as bacteria, fungi and virus.
Microbiological culture:
• It is a method of multiplying microbial organisms by letting them
reproduce in predetermined culture media under controlled laboratory
conditions.
• Microbial cultures are used to determine the type of organism and its
abundance in the sample being tested or both.
Definition:
They are the in-vitro procedures used to detect anti-microbial resistance in
individual bacterial isolates.
 The same can be used for monitoring the emergence and spread of
resistant microorganisms in a population.
 In modern laboratories, bacteria are usually identified by characterization
of the genome : identifying the characteristics of the DNA and RNA of
the sample species
Sensitivity test:
• Also called susceptibility testing
• A laboratory test perform to check the effectiveness of a drug against a
microorganism, to select the best drug regimen that is effective.
 Purpose of culture sensitivity tests:
• To guide the clinician in selecting the best drug for an individual patient.
• To control the use of inappropriate drug in clinical practice.

2. • To accumulate epidemiological information on the resistance of
microorganism of public health importance within the community.
• To reveal the changing trends in the local isolates.
• To overcome the microbial drug resistance.
Why is sensitivity analysis done?
 Unfortunately, many bacteria are resistant to common antibiotics.
 Resistant means that the drug cant kill the bacteria.
 Sensitivity analysis is a useful tool to help quickly determine if bacteria
are resistant to certain drugs.
 Also be used for fungal infections.
Examples of antibiotic resistant infections include:
 A persistent sore throat
 A recurring urinary tract infection(UTI)
 An unresponsive case of pneumonia.
How sensitivity analysis done?
• Sensitivity analysis starts with a bacterial sample.
• The sample is obtained by swabbing the infected area and from any area
that has infection.
• Cultures are taken from blood, Urine, Sputum, inside cervix and a wound.
Steps in analysis:
• The doctor will send the specimen to a licensed laboratory in a special
culture tube for testing.
• There the sample of infective material spread onto a plate of nutrient
substance and the bacteria in the culture will grow and multiply.
• The bacteria will form colonies- large groups of bacteria that will be
exposed to different antibiotics.

3. • With a sufficient population of bacteria grown on the plate in the form of
a lawn, the technicians will perform two main operations:
Identify the species of bacteria:
 This is done with various techniques, including examination of lawn
characteristics-color, texture, growth pattern, etc.
 Bacterial species commonly isolated depends on the location and the
cause of the infection.
 Gram staining, microscopic examination, metabolic requirements and
even DNA sequencing.
Determine the bacterial populations sensitivity to arrange of
antibiotics:
 This can be done by placing small disks of filter paper or the agar
impregnated with various types of antibiotics onto the bacterial lawn.
 The bacteria are allowed to incubate for a specified days and then the late
is examined to see whether the bacterial growth is inhibited or not by the
antibiotics on the each disk.
Methods for microbiological culture sensitivity test:
1.DILUTION METHODS :(Broth and Agar dilution method)
 The broth dilution method involves subjecting a series of
concentrations of anti-microbial agents in a broth environment.
 Micro dilution tests uses about 0.05 to 0.1ml total broth volume
and can be conveniently performed in a micro-titre format.
 Macro dilution testing uses broth volumes at about 1.0ml in
standard test tubes.
 For both these dilution methods, the lowest concentration at which
the isolate is completely inhibited – as evidenced by the absence of
bacterial growth-is recorded as the minimal inhibitory
concentration.

4.  The minimum inhibitory concentration(MIC) is the minimum
concentration of the anti-biotic that will inhibit the particular
isolate .
 The test is only valid only if the positive control shows growth and
the negative control shows no growth.
 A procedure similar to broth dilution is agar dilution.
 Agar dilution method follows the principle of establishing the
lowest concentration of the serially diluted antibiotic concentration
at which bacterial growth is still inhibited.
2. Disc diffusion method:
• Because of convenience, efficiency , the disc diffusion method is most
widely used method.
• A growth medium , usually Mueller-Hinton agar , is first even seeded
throughout the plate with the isolate of interest that has been diluted at a
standard concentration (app 1 to 2x10 colony forming units per ml).
• Then , using a dispenser such as the one pictured, antibiotic-impregnated
discs are placed onto the agar surface . As the bacteria on the lawn grow ,
they are inhibited to varying degrees by the antibiotic diffusing from the
disc.

5. • The test antibiotic immediately begins to diffuse outwards from the discs,
creating a gradient of antibiotic concentration.
• Place the metric ruler across the zone of inhibition, at the widest
diameter, and measure from one edge of the zone to the other edge.
Holding the plate up to the light might help.
• The disc diameter will actually be part of that number . If there is NO
zone at all , report it as 0…..even though the disc itself is around 7mm.
• Zone diameter is reported in millimeters , looked up on the chart , and
result reported as S(sensitive), R(resistant), or I(intermediate).
3.E-Test
• E-Test (AB Bio disk, solna, Sweden) is a commercially available test that
utilizes a plastic test strip impregnated with a gradually decreasing
concentration of particular antibiotic
• The strip also displays a numerical scale that corresponds to the antibiotic
concentration contained therein
• This method provides for a convenient quantitative test of antibiotic
resistance of clinical isolate
• However , separate strip is needed for each antibiotic, and therefore the
cost be high .

6. 4.Genotypic methods
• Although nucleic acid –based detection systems are generally rapid and
sensitive , it is important to remember that the presence of a resistance
gene does not necessarily equate to treatment failure , because resistance
is also dependent on the mode and level of expression of these genes
• Some of the most common molecular techniques utilized for Anti
microbial resistance detection are as follows:
• Polymerization chain reaction:
• It is one of the most commonly used molecular techniques for detecting
certain DNA sequence of interest. This involves several cycles of
denaturation of sample DNA, annealing of specific primers to the target
sequence and the extension of this sequence, in an exponential manner, to
a point ehinch will be visible detectable by gel electrophoresis with the
aid of a DNA-intercalating Chemicals fluorescence under UV light.
DNA hybridization
• This is based on the fact the DNA pyrimidines (cytosine and thymidine)
specifically pair up with purines (guanine and adenine; or uracil for
RNA)
• Therefore, a labelled probe with a know specific sequence can pair up
opened or dentures DNA from the Test sample, as long as their sequence
complement each other.

7. • if this hybridization occurs, the probe labels this with a detectable
radioactive isotope, antigenic substrate, enzyme or chemilumnincenst
compound.
• Whereas if no target sequence is present or the isolate does not have the
specific gene of interest, no attachment of probes will occur, and
therefore no signals will be detected.
• Modifications of the PCR and DNA hybridization with these basic
principles, several modifications have been introduced which further
improve the sensitivity and specificity of the procedure.
• Examples of such development the use of flouresces –labeled
oligonucleotides, the development of molecular bacons, development of
DNA arrays and DNA chips, Among many others
How the method is selected
• Selection of the appropriate method will depend on the intended. Degree
of accuracy, convenience, urgency, bioavailability of sources, availability
of expertise and cost
What are the results for a sensitivity analysis
• The colonies show up as:
• Susceptible, resistance or intermediate
Susceptible :in this case, a clear, circular “ halo” –technically known as a”
plaque “ or zone of inhibition –will. Appear around the antibiotic disc ,
indicating an absence of bacteria. The antibiotic has inhibited their growth
and /or killed them, means that they can’t grow if the drug is present. This.
Indicates an effective antibiotic
• The doctor can use the results to determine the best antibiotic to treat
your infection
Resistant: in the case, the filter paper will have no discernable plaque
around it , meaning that the bacteria can grow even in the presence of
antibiotic. This indicates an ineffective antibiotic.

8. Intermediate :a somewhere cloudy plaque indicates that not all the bacteria
in the area around the disc have been killed. This means that a higher dose of
the antibiotic is needed to prevent the growth
What are the risk of sensitivity analysis
• Few risk are associated with this test blood collection comes with small
risk .
• Rare risk of taking a blood sample include:
• Sightedness or fainting.
• Infection usually prevented by the skin being cleaned before the needle is
inserted.
• Excessive bleeding –bleeding for a long period afterwards may indicate a
more serious bleeding condition and should be reported to the doctor.
• Hematoma –a bruise where blood accumulates under the skin.
• The doctor will advise the patient about the potential risk associated with
the sample.
Conclusion :
• An antibiotic that bacteria, fungus, or another microorganisms shows
resistance to shouldn’t be used to treat infection. Your doctor will decide
which drug is best if several antibiotics are shown to be effective in
killing the microorganisms causing the infection.
Applications :
• To provide a reliable prediction of whether an infection caused by a
bacterial isolated will respond therapeutically to a particular antibiotic
treatment.
• This may be utilized as guidelines for chemotherapy, or at the population
level as indicator of emergence and spread of resistance based on passive
or active surveillance.
Drawbacks :

9. • Because of the required culture time, sensitivity testing may Tai several
days , which is not ideal in critical cases demanding urgency
• Practitioners switch over to anti-bio grams.
Common medias used :
• The culture media are prepared according to the specifications of the USP
, European, pharmacopoeia, British pharmacopoeia and you India
pharmacopoeia.
• The antibiotic media are identified numerically with names assigned by
Grove and Randall seed agar .
• Base agar
• Assay broth
• Yeast beef agar
Quality control :
• Aspects of a quality control include:
• Standardized bacteriaI inoculum size
• Culture conditions growth medium , pH , cation concentration
• Blood and serum supplements and thymidine contents
• Incubation conditions-atmosphere, temperature, duration
• Concentration of anti microbials for testing.