Export

Download as pdf or txt
Download as pdf or txt
You are on page 1of 3

130 DISTRICT LABORATORY PRACTICE IN TROPICAL COUNTRIES

3 Swab the top of the culture bottle and reincu- Note: Recognition of bacteria in a Gram smear,
bate. especially Gram negative organisms, is often
difficult due to background debris.
Contamination of blood cultures
Contamination can occur when an aseptic technique Bacteria that may be found in contaminated bank
is not used at the time the blood is collected or at a blood
later stage in the laboratory when subculturing. These are usually organisms that are capable of
Frequent contaminants of blood cultures include growing at room temperature or below. They
commensal staphylococci, micrococci, and diph- include coliforms, Achromobacter species, and
theroids or contaminants from the environment pseudomonads and less commonly Yersinia species.
such as species of Bacillus or Acinetobacter.
Occasionally in immunocompromised patients,
organisms usually considered ‘contaminants’ may be
pathogenic, especially fungi.
Contamination is indicated when an organism is
7.15 Examination of semen
recovered from only one bottle when it should have
grown in both thioglycollate broth and the diphasic Analysis of semen (seminal fluid) is included in this chapter
culture medium or when a mixed microbial flora is because it is often performed in a microbiology laboratory.
isolated. The values and clinical notes contained in this subunit have
Note: An obligatory anaerobe will be isolated from the thio- been referenced from the WHO laboratory manual for the
glycollate culture only. examination of human semen and semen-cervical mucus inter-
action (see Further information)

Bacteriological investigation of a transfusion When investigating infertility, the basic analysis of


reaction semen (seminal fluid) usually includes:
Severe and often fatal reactions can be caused by  Measurement of volume
the transfusion of contaminated blood. The bac-  Measurement of pH
teriological investigation of a transfusion reaction is  Examination of a wet preparation to estimate the
as follows: percentage of motile spermatozoa and viable
forms and to look for cells and bacteria.
1 Report whether the blood remaining in the unit  Sperm count
of transfused blood shows any visible signs of  Examination of a stained preparation to estimate
being contaminated such as: the percentage of spermatozoa with normal
– appearing unusually dark in colour, morphology.
– containing small clots,
– the plasma appearing red, or unusually Collection and transport of semen
turbid (examine after centrifuging a sample 1 Give the person a clean, dry, leak-proof con-
of the blood). tainer, and request him to collect a specimen of
Note: Haemolysis does not always occur when semen at home following 3–7 days of sexual
blood is contaminated. abstinence.
Note: When a condom is used to collect the
2 Using an aseptic technique, inoculate three fluid, this must be well-washed to remove the
bottles of thioglycollate broth, each with about powder which coats the rubber. It must be dried
4 ml of the well-mixed blood. Mix gently, and completely before being used.
label each bottle with the date and unit number Coitus interruptus: This method of collection should not
of the blood. be used because the first portion of the ejaculate (often
containing the highest concentration of spermatozoa)
Incubate one bottle at 35–37 °C, one at room may be lost. Also the acid pH of vaginal fluid can affect
temperature, and refrigerate the other at 4 °C for sperm motility and the semen may become contaminated
with cells and bacteria.
up to 7 days, examining for growth and subcul-
turing as described under ‘Examining and 2 Ask the person to write his name on the con-
reporting of blood cultures’. tainer, date and time of collection, period of
abstinence, and to deliver the specimen to the
3 Perform a motility test (see subunit 7.3.2) and laboratory within 1 hour after collection.
examine a Gram stained smear of the plasma. During transit to the laboratory, the fluid should

7.14–7.15
M ICROBIOLOGICAL TESTS 131

be kept as near as possible to body temperature. Normal motility: Over 50% of spermatozoa are
This is best achieved by placing the container motile within 60 minutes of ejaculation.
inside a plastic bag and transporting it in a The spermatozoa remain motile for several hours.
pocket in the person’s clothing.
When more than 60% of spermatozoa are non-
motile, examine an eosin preparation to assess
LABORATORY EXAMINATION OF SEMEN whether the spermatozoa are viable or non-viable
Caution: Handle semen with care because it may (see following text).
contain infectious pathogens, e.g. HIV, hepatitis Presence of cells in semen: Report when more than a few leu-
viruses, herpes viruses. cocytes (pus cells) or red cells are present. When pus cells are
seen, examine a Gram stained smear for bacteria.

1 Measure the volume Viability


Normal semen is thick and viscous when ejaculated. – Mix one drop (10–15 l) of semen with 1 drop
It becomes liquefied usually within 60 minutes due of 0.5% eosin solution* on a slide.
to a fibrinolysin in the fluid. When liquefied, measure *Dissolve 0.1 g of eosin in 20 ml of fresh physiological
the volume of fluid in millilitres using a small gradu- saline.
ated cylinder. – After 2 minutes examine the preparation micro-
Normal specimens: Usually 2 ml or more. scopically. Use the 10 objective to focus the
specimen and the 40 objective to count the
2 Measure the pH percentage of viable and non-viable spermato-
– Using a narrow range pH paper, e.g. pH zoa. Viable spermatozoa remain unstained,
6.4–8.0, spread a drop of liquefied semen on the non-viable spermatozoa stain red.
paper. Normal viability: 75% or more of spermatozoa
– After 30 seconds, record the pH. should be viable (unstained). A large proportion of
non-motile but viable spermatozoa may indicate a
pH of normal semen: Should be pH 7.2 or more
structural defect in the flagellum.
within 1 hour of ejaculation. When the pH is over 7.8
this may be due to infection. When the pH is below
4 Perform a sperm count
7.0 and the semen is found to contain no sperm, this
may indicate dysgenesis (failure to develop) of the – Using a graduated tube or small cylinder, dilute
vas deferens, seminal vesicles or epididymis. the semen 1 in 20 as follows:
Fill the tube or cylinder to the 1 ml mark with
3 Estimate the percentage of motile and well-mixed liquefied semen.
viable spermatozoa Add sodium bicarbonate-formalin diluting fluid
Motility (Reagent No. 72) to the 20 ml mark, and mix well.
– Place 1 drop (10–15 l)* of well-mixed liquefied
semen on a slide and cover with a 20  20 mm – Using a Pasteur pipette, fill an Improved
or 22  22 mm cover glass. Neubauer ruled chamber with well-mixed
*1 drop falling from a 21 g needle is equivalent to a
diluted semen. Wait 3–5 minutes for the sper-
volume of 10–15 l. matozoa to settle.

– Focus the specimen using the 10 objective. – Using the 10 objective with the condenser iris
Close the condenser iris sufficiently to give good closed sufficiently to give good contrast, count the
contrast. Ensure the spermatozoa are evenly dis- number of spermatozoa in an area of 2 sq mm,
tributed (if not, re-mix the semen and examine a i.e. 2 large squares.
new preparation). Note: The total area of an Improved Neubauer and a
Bürker ruled chamber is 9 sq mm, i.e. 9 large squares.

– Using the 40 objective, examine several fields


– Calculate the number of spermatozoa in 1 ml of
to assess motility, i.e. whether excellent (rapid
fluid by multiplying the number counted by
and progressive) or weak (slow and non-pro-
100 000.
gressive). Count a total of 100 spermatozoa, and
note out of the hundred how many are motile. Normal count: 20  106 spermatozoa/ml or more.
Record the percentage that are motile and non- Counts less than 20  106/ml are associated with
motile. male sterility.

7.15
132 DISTRICT LABORATORY PRACTICE IN TROPICAL COUNTRIES

5 Estimate the percentage of spermatozoa ● Acrosomal cap absent or abnormally large.


with normal morphology in a stained ● Nucleus contains vacuoles or chromatin is
preparation unevenly distributed.
– Make a thin smear of the liquefied well-mixed ● Two heads.
semen on a slide. While still wet, fix the smear ● Additional residual body, i.e. cytoplasmic droplet.
with 95% v/v ethanol for 5–10 minutes, and
allow to air-dry. Middle piece
● Absent or markedly increased in size.
– Wash the smear with sodium bicarbonate- ● Appears divided (bifurcated).
formalin solution (Reagent No. 72) to remove ● Angled where it meets tail.
any mucus which may be present. Rinse the
smear with several changes of water. Tail
● Absent or markedly reduced in length.
– Cover the smear with dilute (1 in 20) carbol ● Double tail.
fuchsin and allow to stain for 3 minutes. Wash off ● Bent or coiled tail.
the stain with water. Note: Abnormal semen findings should be checked
by examining a further specimen, particularly when
– Counterstain, by covering the smear with dilute the sperm count is low and the spermatozoa appear
(1 in 20) Loeffler’s methylene blue for 2 minutes. non-viable and abnormal. When the abnormalities
Wash off the stain with water. Drain, and allow are present in the second semen, further tests are
the smear to air-dry. indicated in a specialist centre.

Staining results Further information


WHO laboratory manual for the examination of human semen
Nucleus of head . . . . . . . . . . . . . . . . . . . . Dark blue and semen-cervical mucus interaction, 4th edition, 1999. ISBN
Cytoplasm of head . . . . . . . . . . . . . . . . . . . Pale blue 0521645999. Cambridge University Press.
Middle piece and tail . . . . . . . . . . . . . . . . . Pink-red

Alternative stains: Other staining techniques used to stain


spermatozoa include Giemsa and Papanicolaou.
7.16 Antimicrobial
Morphology of spermatozoa susceptibility testing
Examine the preparation for normal and abnormal
spermatozoa using the 40 objective. Use the
100 objective to confirm abnormalities. Count 100 Antimicrobial agents include naturally occurring anti-
spermatozoa and estimate the percentage showing biotics, synthetic derivatives of naturally occurring
normal morphology and the percentage that appear antibiotics (semi-synthetic antibiotics) and chemical
abnormal. antimicrobial compounds (chemotherapeutic
Note: Laboratory staff who have not been trained to report agents). Generally, however, the term ‘antibiotic’ is
stained semen preparations should request the assistance of a used to describe antimicrobial agents (usually anti-
specialist cytology laboratory.
bacterial) that can be used to treat infection.
Normal spermatozoa: Measure 50–70 m in Compared with antibacterial agents, fewer antiviral
length. Each consists of an oval-shaped head (with and antifungal agents have been developed. Many
acrosomal cap) which measures 3–5  2–3 m, a antiviral agents have serious side-effects e.g. those
short middle piece, and a long thin tail (at least used to treat HIV infection.
45 m in length). In normal semen, at least 50% of
spermatozoa should show normal morphology. Antimicrobial activity
Most specimens contain no more than 20% Not all antimicrobials, at the concentration required
abnormal forms. to be effective are completely non-toxic to human
cells. Most, however, show sufficient selective toxicity
Abnormal spermatozoa: The following abnor- to be of value in the treatment of microbial diseases.
malities may be seen: Antibacterial agents can be grouped by their
Head mode of action, i.e. their ability to inhibit the syn-
● Greatly increased or decreased in size. thesis of the cell wall, cell membrane, proteins, and
● Abnormal shape and tapering head (pyriform) nucleic acids of bacteria.

7.15–7.16

You might also like