SPECIMEN COLLECTION,

TRANSPORT, AND
PROCESSING

Once you have completed the experiments in this section, you should be
1. Understand the value of proper specimen collection in microbiology.
2. Understand the basic concepts of proper specimen, transport and processing methods
3. Able to recognize samples that are not suitable and suggest appropriate corrective action

Introduction
In the late 1800s, the first clinical microbiology laboratories were organized to diagnose infectious diseases such as
tuberculosis, typhoid fever, malaria, intestinal parasites, syphilis, gonorrhea, and diphtheria. Between 1860 and
1900, microbiologists such as Pasteur, Koch, and Gram developed the techniques for staining and the use of solid
media for isolation of microorganisms that are still used in clinical laboratories today. Microbiologists continue to
look for the same organisms that these laboratorians did, as well as a whole range of others that have been
discovered, for example, Legionella, viral infections, nontuberculosis acid-fast bacteria, and fungal infections.
Microbiologists work in public health laboratories, hospital laboratories, reference or independent laboratories, and
physician office laboratories (POLs).
Depending on the level of service and type of testing of each facility, in general a microbiologist will perform
one or more of the following functions:
• Cultivation (growth), identification, and antimicrobial susceptibility testing of microorganisms
• Direct detection of infecting organisms by microscopy
• Direct detection of specific products of infecting organisms using chemical, immunologic, or molecular
techniques
• Detection of antibodies produced by the patient in response to an infecting organism (serology)

Discussion
SAMPLING METHODS IN MICROBIOLOGY
The way of sampling depends on the aim of the microbiological study, and must be done at the selected
site with sterile equipment. Although sampling different environments (e.g. throat swab, blood, soil,
sewage, water, etc.) require different methods and equipment, samples always have to be representative.
Sampling must always be done in adequate number of parallels depending on the aim of the research.
Not only sampling, but also the transfer of samples to the laboratory can be a critical point during the
study. Recording details on site will help the interpretation of results later. Take extra care to avoid
contaminating the sample container or the sample.

Sampling for diagnostic purposes

Sampling for diagnostic purposes and the successful diagnosis of pathogenic microbes needs professional
sampling techniques and quick transfer of samples to the laboratory. Different methods are used
for different samples (e.g. tissue, urine, feces, blood, etc.), but the amount of contaminating microbes
in the samples (which possibly mask the original pathogens) must be low. Samples are always put into
sample containers that are adequate for the given sample. Samples must be precisely labelled and
transferred to the laboratory with care.

Sampling from various environments

1. Collection of air samples

Air sampling in the context of microbiological assessment is the collection of airborne microorganisms.
The atmosphere is not a living habitat for microbes, but they can spread through it, and therefore the
atmosphere could act as a conveyor of pathogenic microbes. Studying microbes in the air has not only
hygienic but also economic importance: microbes present in the atmosphere of a factory can be hazardous
to crude materials and products, and also to the production processes. Consequences of using
microbiologically contaminated materials can be serious, therefore checking the quality of the air is a
critical factor in the cosmetics, pharmaceutical and food industries, etc.

There are different ways for sampling the air. The simplest way is the passive, “Koch-type” sedimentation
method (using settle plates), which is adequate to detect well settling microbial particles. Active
methods require impaction devices (Fig. 5). The volume of air for active sample collection depends
on the device being used and on the anticipated concentration of the bioaerosol. With filtration (selecting
adequate pore size filters) or gas washers not only microbes (e.g. fungi, bacteria) but also cell debris
can be detected. The use of impingement (slit sampler) is also widespread. In this case, in a narrow
canal, an air stream is generated and particles are caught by breaking the way of the air.

Where only low concentrations of microbial contaminants are expected, e.g. clean rooms, food production
facilities and operating theatres, generally impaction methods are chosen. In highly contaminated
environments, impaction techniques may 'oversample' even in short timescales and impingement or
filter samples are more appropriate. With the strict adherence to manufacturer's flow rates, sampling
periods, culture media used and device placement, most techniques should yield comparable results,
which are normally expressed in CFU/m³ of air (CFU= colony forming unit).

2. Collection of soil samples

Soil tests measure the microbial composition and activity of different soil horizons. The physicochemical
characteristics of soil influence the rate of biomass production and the activity and composition
of microorganisms. Seasonal changes in soil moisture, soil temperature and carbon input from crop
roots, rhizodeposition (i.e. root exudates, mucilage, sloughed cells), and crop residues can have a large
effect on soil microorganisms, which, in turn, affect the ability of the soil to supply nutrients to plants
through the turnover of soil organic matter. Therefore, the collection of representative soil samples is extremely
important and sampling should always be performed taking into account the above-mentioned
heterogeneity associated with many soil types.
Sampling can be performed aerobically or anaerobically with sterile sampling equipment.

3. Collection of water samples

Sampling natural waters representatively is one of the biggest challenges to overcome. In flowing
waters, the trail effect, in stagnant waters, stratification make representative sampling difficult. Moreover,
coastal current and drifting also must be considered. Surface water samples are taken from 10 cm depth
by submerging sterile sampling bottles. For the collection of samples from deeper zones, special
equipment is used. For collecting surface phytoplankton samples, a clean plastic bottle is adequate.
For sampling water-conduit, sampling taps are used, where the water must run for 2-5 min
before taking a sample. The rules for microbiological investigations and sampling are set in the EN
ISO 19458:2006 standard.

4. Sampling the surface of objects

Objects and surfaces can be sampled easily using a sterile cotton swab or with special sampling
equipment (e.g. contact slide).

5. Hygienic control of the hands of operators

In many cases (e.g. clean spaces, in pharmaceutical and food production) low germ counts and environments free of
pathogenic microbes are essential. This also includes the control of personnel. In such
cases usually the palm and finger skin surfaces are sampled with cotton swabs or using contact sampling. The
efficacy of hand hygiene agents can also be tested using these methods.

QUESTIONS FOR RESEARCH:

1. To assist hospital personnel in collecting the highest quality specimen, what should the laboratory do?

2. What is the goal of the specimen collector when collecting specimens for culture?
3. Why does a fecal sample for culture need to be collected in a leak-proof, nonsterile container?
4. To ensure collection of a good specimen for culture, how should specimen collection instructions be given
to the patient?
5. Why is a clean-catch midstream urine used for a urine culture as opposed to a clean-catch urine?
6. What is the best way to minimize the amount of upper respiratory flora in a sputum specimen?
7. When detecting gastrointestinal pathogens, what is the specimen of choice for?
8. The laboratory needs specific information regarding the patient and the specimen to perform a quality
laboratory analysis.What can be a critical weak link in the specimen management process?
9. What should the laboratory requisition form provide?
10. If a test is not considered appropriate for the specimen yet the test was still requested, what should be the
proper solution?
11. What should be the safe method of transporting aspirated material?
12. What is the primary goal in the transportation of specimens to the laboratory?
13. If transport of the specimen is delayed, what will you do so that the integrity of the specimen can be
maintained?
14. A night tech is working in microbiology when a cerebrospinal fluid (CSF) specimen comes in. Almost
simultaneously,the tech is called to the emergency department to draw blood on seriously injured car accident
victims.How would the tech store the CSF until time permits to work on the CSF specimen?
15. Enumerate types of specimens that can use preservatives to maintain them until they can be delivered to the
laboratory. Expalin preservation method of each specimens.
 16. Why should specimens such as blood,bone marrow,and synovium are mixed with anticoagulants right after
collection?
17. In certain instances, it is desirable for specimens to be inoculated directly onto culture media. What
pathogen should be placed onto a commercial transport system called a JEMBEC system.?
18. How does the Department of Transportation (DOT) define an infectious substance?
19. Give the four levels that represent a scheme for prioritizing the handling of specimens.
20. You are a lab technician in the laboratory, and during your shift you received a specimen that was rejected.
You then called the physician and told him that the specimen was unacceptable and the reason why.The physician
insisted that the culture needs to be performed on this unacceptable culture.What should the you do?
21. What are the specimen sources which are not recommended for direct microscopic examinations?
22. What method is used to concentrate specimens to increase recovery of pathogens in the microbiology
laboratory?
23. What work practices have microbiologists incorporated to keep laboratory testing cost effective while
providing optimum patient care?
24. You are a laboratory technician in the microbiology laboratory, and to help the microbiologist report
microbiology results to the physician in a timely fashion to ensure the appropriate treatment, what would you do?
25. For each of the following specific sites, describe specific collection requirements (container, patient
preparation, special instructions, transport, storage, primary plating media and direct examination):
a. Throat g. Body fluids (amniotic, abdominal, ascites etc.)
b. Nasopharyngeal h. Gastric aspirate
c. Sputum i. Cerebrospinal fluid
d. Stool j. Urine (clean catch, catheterized, suprapubic)
e. Wound k. Genital (male urethral, female vaginal and cervical)
f. Blood l. Hair, Nails, or Skin Scrapings
26. Name important colonial characteristics observed in the identification of bacteria.
27. What are the aspects of quality control that are applied in Microbiology Laboratory?