Laboratory Exercise 5

Tube Agglutination Test

The Widal Test
Answers to Questions
1. What is the exact meaning of the titer of a serum

It is defined as the lowest dilution of the sample that retains a detectable activity (Murray et al.).
The titer of a serum is defined as the highest dilution of serum at which the antigen undergoes
agglutination (Parija, 2012).

2. Differentiate between the following:

Serum: the liquid portion of blood after the clotting factors have been removed and an important
site of antibodies
Antiserum: serum containing antibodies raised against a specific antigen
Antitoxin: protective antibodies that inactivates soluble toxic protein products
(Bauman et al., and Selamawit Debebe,)

3. How would you prepare antiserum for an organism such as E. coli?

E. coli has O, H and K or G antigens; therefore an antiserum should contain antibodies for these
antigens in order to indentify the strains E. coli (Parija, 2012).

4. Indicate the type of antigen (soluble, protein, red blood cells, or bacteria) that is used for
each of the following serological tests:

Agglutination: Specific antibodies or agglutinin bind to surface antigens and caouses visible
clump formation.
Precipitation: Antibody (precipitins) interacts with the soluble antigen in the presence of
electrolyte at a specified pH and temperature to produce a precipitate
Hemolysis: Lysis of RBC observed when using specific antisera
(Parija, 2012)

5. For which one of the above tests is complement necessary?

Hemolysis (Parija, 2012).

Laboratory Exercise 6
 Bactericidal Power of Normal Serum

Answers to Questions

1. What is/are responsible for the bactericidal action of the serum?

Heat stable substances of beta-lysin and are independent of complement (C), and heat-
labile substances composed of beta-lysin and are dependent of complement (C) and antibodies
(ABs) (Tekman, 2017).

2. What is the difference between the components of unheated and heated serum?

Unheated serum contains proteins, antibodies, complement, growth factors, hormones,

amino acids, glucose, trypsin inhibitors, and lipids. Heat-labile components (such as
complement, growth factors, vitamins, amino acids, and hormones) are destroyed when heated to
56oC

3. Compare the bactericidal activity of heated and unheated serum.

When heated, immunoglobulin aggregation occurs and anti-complementary activities of

heat-stable and heat-labile substances are observed. This results to inhibition of phagocytosis,
and antibody-dependent, cell-mediated cytotoxicity, impaired macrophage migration, diminished
platelet aggregation, and complement fixation which means heated serum has lower bactericidal
activity (Soltis et al., 1978).
 Laboratory Exercise 5
Rapid Plasma Reagin (RPR) Test for Syphilis

Answers to Questions

1. What does RPR determine?

It measures IgM and IgG antibodies to lipoidal material released from damaged host cells
as well as lipoprotein-like material, and possibly cardiolipin from the treponemes (Larsen et al.,
1996).

2. What are the advantages of RPR over VDRL test?

RPR is prepared from a modified VDRL antigen suspensions, it contains ethyl chloride to
eliminated the need to heat inactivate serum, ethyldiaminetetraacetic acid (EDTA) to stabilize the
suspension and finely divided charcoal powder as a visualizing agent, and as compared to VDRL
test this can be performed in the field, and it has improved reaction visibility.

3. What component of the RPR antigen is involved with a reactive patients sample?

The lipid particles (cardiolipin) of the antigen combine with the antilipoidal antibodies
that are present in the sample. These antibodies are produced as a consequence of syphilis and
other treponemal diseases, and in response to nontreponemal diseases (Larsen et al., 1996).

4. Why are charcoal particles incorporated into the RPR test?

They coagglutinate with the antibodies and show up as black clumps against a white card
(Larsen et al., 1996).

5. Why does a positive RPR not necessarily indicate syphilis infection?

The antibodies (antilipoidal antibodies) that are taken into account are produced by not
only as a consequence of syphilis but as well as in response to other nontreponemal diseases of
an acute and chronic nature in which tissue damage occurs. Therefore, it can test positive, but
because of nontreponemal diseases (Larsen et al., 1996).

Laboratory Exercise No. 8

Microbiology of the Urinary and Genital Tracts:
Urine Culture Techniques
1. Why is aseptic urine collection important when cultures are ordered?

The use of aseptic technique is important to avoid contaminations that may interfere with the
accurate identification of bacteria present in the urinary tract (Brooks et al., 2013)

2. If you counted 20 colonies from a 0.01-ml inoculum of a 1:10 dilution of urine, how many
organisms per milliliter of specimen would you report? Is this number significant?

Represented in milliliter, the number of colonies would be 20,000/ml. This result represents the
absence of infection, since the presence of infection is represented by a colony count of 100,000
colonies per ml, however lower colony counts may also dictate infection (AACC, 2016).

3. What can you learn from visual inspection of a urine specimen?

Visual inspections can be used to diagnose a disease, and infection is associated with a cloudy
(presence of other substances such as leukocytes, bacteria, or epithelial cells), colored (due to
concentration of solutes, or presence of blood), and also unusual odor (Brooks et al., 2013).

4. How are UTIs acquired/transmitted?

Urinary tract infections may be transmitted by improper personal hygiene, and contamination
during medical procedures, and are often caused by opportunistic pathogens (Tortora et al.,
2016).

5. Explain why E. coli is frequently implicated in cystitis in females.

Cystitis are more often implicated in female because the anus is closer to their urethra, and they
have a shorter urethra, which means bacteria may be able to get into the bladder more easily
(Tortora et al., 2016).
 Laboratory Exercise No. 9
Culture and Microscopic Examination of Fungi

1. Define budding and hyphae.

Hyphae is a long filament of fungal cells or actinomycetes, and budding is an asexual

reproduction that begins as a protuberance from the parent cell and grows to become a daughter
cell (Tortora et al., 2016)

2. Why do yeasts generally have to be cultured for longer periods than most bacteria?

Fungi are eukaryotic protists which are more complex and differ in many ways with bacteria
such as in the time it needs to culture fungi. Bacteria reproduce rapidly through binary fission,
while yeasts have a lengthier generation time (Kumar, 2012).

3. What are some similarities and differences of the yeasts that were cultured from your
mouth?

N/A

4. What is the difference between vegetative and aerial mycelia?

A mycelium is constituted by a tangled mass of hyphae that forms in molds or filamentous fungi.
A vegetative mycelium is a hypha that penetrates the supporting medium and absorb nutrients,
while aerial hyphae project the surface of the mycelium and bear the reproductive structures of
the mold which produces asexual pores (Kumar, 2012).

5. Can bacteriological media be used for the cultivation of molds? Explain your answer.

Yes. Most fungi occur in nature and grow readily on simple sources of nitrogen and
carbohydrate, so bacteriological media that contain glucose and modified peptone with a neutral
pH may also be used to cultivate molds.
 Laboratory Exercise No. 10
Bacterial Mutation

1. How would you describe a streptomycin-dependent mutant bacterium?

It is a mutant bacterium that harbors resistance to streptomycin which is due to the

substitution of a single amino acid (rpsL gene encoding the S12 ribosomal protein). It requires
the binding of streptomycin to its protein to function properly during protein synthesis (Gregory
et al., 2001).

2. How can bacterial mutations occur?

This may result from the exposure of the bacterium to radiation or chemicals (antibiotics)
that cause changes in the bacterial DNA (Kumar, 2012).

3. What is a Mutagen? Give several examples.

Mutagens are environmental agents such as chemicals or radiation. Chemical mutagens

include nitrous acid, nucleoside analogue, benzopyrene and aflatoxin; radiations may be X-rays,
gamma-rays or UV light (Tortora et al., 2016).

4. What methods could be used to increase the frequency of the appearance of mutant
Staphylococcus aureus.

Antimicrobial use and misuse, and factors that enhance its transmission may be increase
the frequency of mutant S. aureus.