MCB 204: Microbiological Techniques II

COLLECTION, HANDLING AND PROCESSING OF SPECIMEN

Objectives

1. What is sample collection

2. How are the samples collected
3. What are the methods of processing these samples
4. Transportation of the Samples

Collection of Specimen

Specimen collection defined as the collection of the specimen for the purposes of diagnosis,
treatment and recovery. Specimens are collected to aid in the screening and diagnosing of patient
health problems, directing treatment and monitoring the effectiveness of treatments. The most
commonly collected specimens are blood, urine, stool, and sputum.

Significance of Specimen Collection

Specimen collection in microbiology is done in order to isolate and identify the causative agent
of a disease, without successful isolation and identification of microorganisms none of disease
can be precisely diagnosed nor treated properly. This forms the back bone of all the investigative
procedures. The goal of microbiologic evaluation is to provide accurate, clinically pertinent
results in a timely manner. The quality of the specimens submitted to the microbiology
laboratory is critical for optimal specimen evaluation. The general techniques of specimen
collection and handling that have been established both to maximize the yield of organisms and
isolate relevant pathogens from specimens obtained from different body.
Steps to consider when Collecting Specimen
1. What to collect? (Type of specimen).
2. How to collect (Procedure).
3. When to collect specimen (Best time of specimen collection)
4. Use the proper aseptic collection technique.
5. Need for any preservative or transport medium.
6. Labeling of the specimen container.
General Rules for Collection of Specimen

1. Use aseptic technique for specimen collection.

2. Start collection of specimens for all cultures before starting an Antibiotic treatment.
3. Prevent contamination from normal flora.
4. Specimen should be adequate in amount.
5. Need for transport medium or any preservative.
6. Collection should be done in a sterile, leak proof, properly labeled tightly closed
container.
7. Specimen should be accompanied with information paper/request form. Information
paper has name, age, sex, address of patient, type of specimen, investigation required,
date and time of specimen collection, name and signature of the doctor.
8. Prompt processing of specimen and transportation to the laboratory after collection.

Type of specimen

The correct type of specimen to collect will depend on the pathogens to be isolated. Example:
Cervical (from the cervix) not vaginal swab is required for the most successful isolation of N.
gonorrhoeae from a woman.

Sputum not saliva is essential for the isolation of respiratory pathogens e.g Mycobacterium
tuberculosis.

Time of Specimen Collection

Best time to collect urine and sputum is early in the morning when patient wakes up (because
microorganisms have had the opportunity to multiply for several hours). Example: For collection
of blood for culture best time is when the temperature of patient is rising (because at that time
maximum number of microorganisms are present in the blood).

Labeling Specimens

1.The date.

2.Timeof collection.

3.The patient’s name, number, ward or health centre.

Use of Preservative/Transport Medium

When facilities are not available to perform the desired tests at the place of collection or
laboratory is located far away. Then we use some suitable preservative or transport culture
medium in order to keep suspected microorganisms alive and also to suppress growth of
unwanted organisms. Transport media are basically buffer solutions that contain carbohydrate,
peptones, along with other nutritional elements (excluding growing factors) that are designed to
protect the health of bacteria during transport, without allowing for their multiplicity. The main
goal of utilization of the medium is to keep the sample as close to its original condition as is
possible. Examples: Cary and Blair Medium

Cary and Blair Medium semi-solid, white-colored transport medium for feces that may
contain Salmonella, Shigella, Vibrio, or Campylobacter.

Amies medium

1. Amies medium with charcoal: Charcoal helps eliminate metabolic products of bacterial
growth, which may be especially useful in isolating fastidious organisms. However, it is
suggested that other pathogens, like Campylobacter can also survive in such a medium.

2. Amies medium without charcoal: These are ideal for

isolating Mycoplasma and Ureaplasma.

Stuarts medium

Commonly used for transporting specimens suspected of having gonococci. Also used for
transporting throat, wound, and skin swabs that may contain fastidious organisms.

Venkatraman Ramakrishnan (VR) medium

VR medium was used to transport feces from suspected cholera patients. It is no longer used
nowadays. Cary Blair media is the ideal and WHO-recommended media for transporting Vibrio
cholerae.

Alkaline Peptone Water

Alkaline peptone water (APW) is used to transport Vibrio cholerae only when the Cary-Blair
medium is unavailable, and subculture can be done within 6 hours of sample collection.
Sach’s buffered glycerol saline

Used to transport feces from patients suspected of suffering from bacillary dysentery.

Viral Transport Medium

Viral Transport Medium (VTM) is ideal for transporting sample for the diagnosis of viral
infections. Ocular, respiratory, and tissue swabs can be submitted in this medium. Fluid samples
such as tracheal wash specimens or peritoneal fluid should be submitted in sterile vials,
preventing desiccation. In the absence of a viral transport medium, submit swabs in sterile,
sealed vials with several drops of saline added to avoid desiccation. Cotton, plastic, wood-
handled, dacron, and other synthetic swabs are acceptable. Calcium alginate swabs should be
avoided. Bacterial transport media are not appropriate for recovering viral pathogens.

Anaerobic Transport Medium (ATM)

Anaerobic transport medium is a mineral salt base semi-solid media with reducing agents
designed as a holding medium for maintaining the viability of anaerobic bacteria It contains
buffered mineral salts in a semi-solid media with sodium thioglycolate and cysteine added to
provide a reduced environment. Resazurin may also be added as a redox indicator to reveal
exposure to oxygen by turning pink. It provides an environment that maintains most
microorganisms’ viability without significant multiplication and allows for dilution of inhibitors
present in clinical material. Examples include thioglycollate broth.

Containers used

There is a great variety of containers in which microbiological samples can be collected, with a
swabs can be made of different materials 2: dacron, rayon or nylon, cotton (not recommended
for Chlamydia spp., Bordetella spp. and Neisseria gonorrhoeae), calcium alginate (may inhibit
PCR techniques and be toxic to viruses) and may have the smooth absorption surface or be
flocked; they can be used dry or use Amies transport medium in gel or liquid (recommended for
automated work stations).4 The handle should be made of aluminium or plastic (rigid or flexible).
The size and shape of the swab will vary depending on the anatomical location and the type of
sample to be taken.
There are special transport systems, such as bags, vials or tubes with an anaerobic atmosphere,
micro-haematocrit capillary tubes (Trypanosoma spp.), brushes in liquid transport medium for
the papilloma virus, transport medium for universal virus (also valid
for Chlamydia spp., Ureaplasma spp., and Mycoplasma spp.) or sterile tubes with fixatives for
parasites or with preservative such as boric acid-sodium formate for the culture of urine. Blood
culture bottles, lysis-centrifugation bottles, tubes and bottles with screw closure, vacuum tubes
with additives (EDTA, citrate) or separating gel (for serum samples), sterile petri dishes and
syringes for obtaining aspirates can also be used.
ENUMERATION OF MICROORGANISMS.

Enumeration of bacteria is defined as the process of determining the number of bacteria in a

given sample. There are numerous reasons why researchers have to calculate the number of
bacteria or compare the growing number of these microbes under certain specific conditions. It’s
mainly essential and a part of routine work in food, water, and dairy microbiology labs. The
knowledge of the numbers of these microorganisms in our food, including milk, curd, buttermilk,
and water, help the labs to determine if the prepared food or available water is hygienic or not for
consumption. For unicellular microorganisms, such as bacteria, the reproduction of the cell
reproduces the entire organism. Therefore, microbial growth is essentially synonymous with
microbial reproduction. To determine rates of microbial growth and death, it is necessary to
enumerate microorganisms, that is, to determine their numbers. It is also often essential to
determine the number of microorganisms in a given sample. For example, the ability to
determine the safety of many foods and drugs depends on knowing the levels of microorganisms
in those products. A variety of methods has been developed for the enumeration of microbes.
These methods measure cell numbers, cell mass, or cell constituents that are proportional to cell
number. The four general approaches used for estimating the sizes of microbial populations are
direct and indirect counts of cells and direct and indirect measurements of microbial biomass.

Methods for counting microorganisms

In some cases, it is necessary to know the total number of (both living and dead)
microorganisms. For example, in endotoxin (pyrogen) testing, dead and alive cells induce fever
when injected into the body. However, in many cases, the number or concentration of living cells
is required. Total Count Technique: Total count is a counting procedure enumerating both living
and dead cells.

Direct Microscopic count

Direct microscopic counts are possible using special slides known as counting chambers,
consisting of a ruled slide and a coverslip. Direct microscopic counts are performed by spreading
a measured volume of sample over a known area of a slide, counting representative microscopic
fields, and relating the averages back to the appropriate volume-area factors. Specially
constructed counting chambers, such as the Petroff-Hauser and Levy counting chambers,
simplify the direct counting procedure because they are made with depressions in which a known
volume overlies an area that is ruled into squares. The ability to count a defined area and convert
the numbers observed directly to volume makes the direct enumeration procedure relatively easy.
It is constructed so that the coverslip, slide, and ruled lines delimit a known volume. The number
of bacteria in a small known volume is microscopically counted, and the number of bacteria in
the larger original sample is determined by extrapolation. Dead cells cannot be distinguished
from living ones. Only dense suspensions can be counted. This special slide is accurately ruled
into squares that are 1/400 mm² in the area; a glass coverslip rests 1/50 mm above the slide so
that the volume over a square is 1/20,000 mm² i.e., 1/20, 000, 000 cm². If, for example, an
average of five bacteria is present in each ruled square, there are 5 x 20,000,000 or 10 8 bacteria
per milliliter. A suspension of unstained bacteria can be counted in the chamber using a phase-
contrast microscope.

The formula used for the direct microscopic count is:

Total cells counted x 2.0×107 x dilution factor/ number of small squares counted =
cells/ml

Advantages of direct microscopic count

 Direct counting procedures are rapid, simple, and easy.

 You can observe the morphology of the bacteria while counting the numbers.

 After dilutions, even dense suspensions can also be counted using the technique.

Limitations of direct microscopic count

 They do not discriminate between living and dead cells.

 It cannot be used to count viable cells.

 You can’t observe small cells, so they can be missed during counting.

 This method cannot be used with cell suspensions of low density, i.e., <107 cells per ml.
Turbidity Method

Turbidity measurements are the most common means of estimating the total number of bacteria
present in a sample. Measuring the turbidity using a spectrophotometer or colorimeter and
reading the concentration from a calibration plot is a simple means of standardizing cell
suspensions for inocula in antibiotic assays or other tests of antimicrobial chemicals. When we
mix the bacteria growing in a liquid medium, the culture appears turbid. This is because a
bacterial culture acts as a colloidal suspension that blocks and reflects light passing through the
culture. Within limits, the light absorbed by the bacterial suspension will be directly proportional
to the concentration of cells in the culture. By measuring the amount of light absorbed by a
bacterial suspension, one can estimate and compare the number of bacteria present. The
instrument used to measure turbidity is a spectrophotometer. It consists of a light source, a filter
that allows only a single wavelength of light to pass through, the sample tube containing the
bacterial suspension, and a photocell that compares the amount of light coming through the tube
with the total light entering the tube.
The ability of the culture to block the light can be expressed as either percentage of light
transmitted through the tube or amount of light absorbed in the tube.

The percentage of light transmitted is inversely proportional to the bacterial concentration. (The
greater the percent transmittance, the lower the number of bacteria.) The absorbance (or optical
density) is directly proportional to the cell concentration. (The greater the absorbance, the greater
the number of bacteria.) Turbidimetric measurement is often correlated with some other method
of cell count, such as the direct microscopic method or the plate count. In this way, turbidity can
be used as an indirect measurement of the cell count. For example:

1. Several dilutions can be made of a bacterial stock.

2. A Petroff-Hausser counter can then be used to perform a direct microscopic count on

each dilution.

3. Then, a spectrophotometer can be used to measure the absorbance of each dilution tube.

4. A standard curve comparing absorbance to the number of bacteria can be made by

plotting absorbance versus the number of bacteria per cc.

Figure 1: A standard curve plotting the number of bacteria per cc versus absorbance.

5. Once the standard curve is completed, any dilution tube of that organism can be placed in
a spectrophotometer and its absorbance read. Once the absorbance is determined, the
standard curve can be used to determine the corresponding number of bacteria per cc.
 Figure 2: Using a standard curve to determine the number of bacteria per cc in a sample by
measuring the sample’s absorbance.

Advantages of turbidimetric measurement

 It’s a faster procedure than the standard plate count technique.

 It can be done without spoiling the samples.

Limitations of turbidimetric measurement

 For the estimation, it requires a high amount of bacterial cells—about 100 million cells
per milliliter.

 If the mass of bacteria increases more than normal in the sample, they mask the light
passed through the cuvette, resulting in inaccurate calculations.

Direct Count Using Fluorescent Dyes

Fluorescent dyes are becoming more used in recent years for a variety of procedures, one of
which is bacterial counts. These dyes can be employed to stain all species, a particular species of
interest in an environmental sample or even a specific component of cells. The most widely used
fluorescent dye for counting the number of bacterial cells is acridine orange which stains both
living and dead cells by interacting with DNA and protein components of cells. The stained cells
fluoresce orange when excited near ultraviolet light. This stain is particularly useful for
determining the total number of microorganisms in samples, such as soil and water, where the
co-existence of metabolically diverse populations precludes establishing conditions for the
simultaneous enumeration of microbial populations by viable count procedures. The procedure is
widely used in marine microbiology where population levels are often low and where viable
plate counts are known to severely underestimate total number of bacteria. Typically, the viable
count is less than 1% of the direct count for marine samples. In this procedure the bacteria in a
known volume of sample are stained with acridine orange and the sample is then filtered through
a 0.22 µm filter. The bacteria are trapped on the filter that is then examined under a fluorescence
microscope. The bacteria in a defined area of the filter are counted and the concentration in the
original sample is then calculated. Other fluorescent dyes that are also gaining popularity are
Cyanoditolyl Tetrazolium Chloride (CTC), Auramine and Rhodamine. CTC binds to respiration
proteins in the cell and thus can demonstrate live cells. Auramine and rhodamine bind to cell
wall of Mycobacteria and emit bright yellow or orange color under a fluorescent microscope.
These latter stains are gradually replacing the acid-fast stain.

Indirect Count of Cells

Microorganisms in a sample are diluted or concentrated and grown on a suitable medium; the
development of growing microorganisms (for example, colony formation on agar plates) is then
used to estimate the numbers of microorganisms in the original sample.

Viable Count

The most common procedure for the enumeration of bacteria is the viable plate count. A viable
cell is defined as one that can divide and form offspring. In this method, serial dilutions of a
sample containing viable microorganisms are plated onto a suitable growth medium. The
suspension is either spread onto the surface of agar plates (spread plate method), or is mixed with
molten agar, poured into plates, and allowed to solidify (pour plate method). The plates are then
incubated under conditions that permit microbial reproduction so that colonies develop that can
be seen without the aid of a microscope. It is assumed that each bacterial colony arises from an
individual cell that has undergone cell division. Therefore, by counting the number of colonies
and accounting for the dilution factor, the number of bacteria in the original sample can be
determined. There are several drawbacks to the viable count method. The major disadvantage is
that it is selective and therefore biased. The nature of the growth conditions, including the
composition and pH of the medium used as well as the conditions such as temperature,
determines which bacteria in a mixed population can grow. Since there is no universal set of
conditions that permits the growth of all microorganisms, it is impossible to enumerate all
microorganisms by viable plating. This same disadvantage, however, becomes advantageous
when one is interested in only a specific microbial population. For example, we can design
selective procedures for the enumeration of coliforms and other physiologically defined
microbial groups. The viable count is an estimate of the number of cells. Because some
organisms exist as pairs or groups and because mixing and shaking of the sample does not
always separate all the cells, we actually get a count of the "colony forming units" (CFU). One
cell or group of cells will produce one colony, therefore when we record results for a viable
count, it is customary to record the results as colony forming units per ml (cfu/ml) or per gram
(cfu/g) of test material. Because we generally have no idea of how many bacteria are in a sample,
it is almost always necessary to prepare a dilution series to ensure that we obtain a dilution
containing a reasonable number of bacteria to count. Dilutions in the range 10-1 (1/10) to 10-8
(1/100,000,000) are generally used, although with particular types of samples the range of
dilutions can be restricted. For example, for water that is not turbid, the maximal dilution needed
is 10-6 because we know that if there were 107 or more bacteria per milliliter, the water would be
turbid. The formular for determining the viable cell count is represented as:

The number of CFUs per ml of sample = The number of colonies (30-300 plate) X The dilution
factor of the plate counted

Advantage of viable count method

This method is used routinely and with satisfactory results for estimating bacterial populations in
milk, water, foods, and many other materials.

1. It is easy to perform and can be adapted to measuring populations of any magnitude.

2. It is a sensitive method (theoretically, a single viable cell can be detected). For example,
if a specimen contains as few as one bacterium per ml, one colony should develop upon
the plating of 1 ml.
 3. It allows for inspection and identification of the organism counted.

Limitation of viable count technique

1. Only living cells develop colonies

2. Clumps or chains of cells develop into a single colony

3. Colonies develop only from those organisms for which the cultural conditions are
suitable for growth.

The Most Probable Number (MPN)

The most probable number procedure dates back to the earliest days of microbiology. However,
it is still widely used in sanitary bacteriology to estimate numbers of coliforms in water, milk,
and other foods. Coliforms are bacteria that reside in the intestine of warm-blooded mammals
and are regularly excreted in the feces. They are Gram negative rods belonging to the
Enterobacteriaceae family, ferment lactose and produce gas. Not all members of
Enterobacteriaceae are coliforms. The MPN procedure is a statistical method based upon the
probability theory. Samples are serially diluted to the point of extinction, that is, to a point where
there are no more viable microorganisms. To detect the end point, multiple serial dilutions are
inoculated into a suitable growth medium, and the development of some recognizable
characteristic, such as acid production or turbidity, is used to indicate growth (the presence of at
least one viable microorganism in the diluted sample). The pattern of positive tests (growth) in
the replicates and statistical probability tables are used to determine the concentration (most
probable number) of bacteria in the original sample. Statistical MPN tables are available for
replicates of 3, 5, and 10 tubes of each dilution. The more replicate tubes used, the greater the
precision of the estimate of the size of the bacterial population.

Direct Measurement of Microbial Biomass

Cell mass is determined directly by weighing whole cells; biomass can be correlated with cell
numbers by reference to a standard curve. Wet weight or dry weight of bacteria may be used for
estimation of cell numbers.
Indirect Measurement of Microbial Biomass

Microbial biomass is estimated by measuring relatively constant biochemical components of

microbial cells, such as protein, ATP, lipopolysaccharides, peptidoglycan, and chlorophyll;
biomass can also be indirectly estimated by measured turbidity that can then be correlated with
cell numbers by reference to a standard curve. Various procedures based on the detection of
specific microbial macromolecules or metabolic products can be used to estimate numbers of
microorganisms. For example, peptidoglycan can be quantified, and because this biochemical
occurs exclusively in the cell wall of bacteria, the concentration of peptidoglycan can be used to
estimate bacterial numbers. Such biochemical approaches for determining bacterial numbers
depend on the development of analytical chemical procedures for quantifying the particular
biochemical and determining what proportion of bacterial cell is composed of the specific
biochemical constituent.

Rapid Methods

Rapid methods enumerate viable microorganisms, usually bacteria and yeasts, within a matter of
hours and eliminate traditional methods’ 24-48hour (or longer) incubation periods. They employ
various means for indirect detection of living cells. Rapid methods are fast, readily automated,
and eliminate the need for numerous Petri dishes and incubators but may require the purchase of
expensive equipment. Detection limits may vary based on the equipment types, test types, and
manufacturers.

Epifluorescent Technique

It uses fluorescent dyes that either exhibit different colors in living and dead cells (e.g., acridine
orange) or appear colorless outside the cell but become fluorescent when absorbed and subjected
to cellular metabolism (e.g., fluorescein diacetate).

ATP Testing

Living cells generate adenosine triphosphate (ATP) that can readily be detected by enzyme
assays, e.g., luciferin emits light when exposed to firefly luciferase in the presence of ATP; light
emission can be measured and related to bacterial concentration.
 Impedance Technique

The resistance, capacitance, or impedance of a culture medium changes due to bacterial or yeast
growth and metabolism, and these electrical properties vary in proportion to cell concentration.

Manometric Technique

Manometric techniques are appropriate for monitoring the growth of organisms that consume or
produce significant quantities of gas during their metabolism, e.g., yeasts or molds producing
carbon dioxide from fermentation.

Traditional and rapid methods of enumerating microorganisms

Viable counts Rapid methods (indirect

Total counts (traditional method)
(traditional method) viable counts)

Direct microscopic counting (using Helber or Epifluorescence often coupled

Pour plate method
haemocytometer counting chambers) with image analysis

Surface spread or spread

Turbidity methods ATP testing
plate method

Membrane filter method Dry weight determination Impedance method

MPN Method Nitrogen, protein, or nucleic acid determinations Manometric methods