Temperature Requirements *EQ*

 Psychrophilic bacteria are cold loving and grow best at 15-20 oC

 Mesophyllic bacteria grow best at 30-37oC. This group includes most pathogenic
bacteria. Thermophilic bacteria are heat loving and most grow best at 50-60 oC. Stool
cultures looking for enteric campylobacters are normally incubated at 42 oC

Morphological Characteristics

 Morphology (the science of shape) can be studied in two ways:

a) Staining characteristics - e.g., staining a smear made on a slide by a selective
dye or series of dyes, e.g., Gram stain.
b) Cultural characteristics - colony growth characteristics and chemical reactions.

Biochemical Activities

 All micro-organisms react with biochemical reagents in a specific manner - e.g., E.

coli will react with lactose and ferment it.

Hemolysis

 When using blood agar plates, certain bacteria hemolyse the blood in characteristic
ways.
a) Alpha: Alpha (α)-hemolytic colonies are surrounded by a zone of greenish
discoloration. This is known as incomplete hemolysis
b) Beta: (β)-hemolysis results in the complete lysis of RBCs. β-hemolytic colonies are
surrounded by a clear zone.
c) Gamma: (γ)- hemolytic colonies are non-hemolytic
d) Alpha prime: (α’)-hemolysis, which may be confused with β-hemolysis, has a zone of
α-hemolysis surrounded by β-hemolysis.

Pathogenicity

 This applies to the disease causing bacteria - e.g., an Elek plate is set up in the case
of Corynebacterium diphtheria to discover if toxin (poison) is being formed by the
organism.
Antigenic Structure

 This quality of micro-organisms is studied by a number of different methods. An

antigen is a substance (in micro-organisms usually a toxin) that reacts in the animal
body to create an immune response by the animal (i.e., the animal produces
antibodies).
 For humans we use SERUM, serological studies.

As you can see, micro-organisms have a great number of features which one must take
into account when isolating and identifying an organism causing a disease condition.
The area which we will concentrate on is that of morphological characteristics - i.e.,
staining and cultural methods. We will also consider oxygen requirements, nutritional
requirements, and temperature.

The types of specimens received routinely in the microbiology laboratory, for bacterial,
fungal and specific cultural procedures include:
1. Autopsy material
2. Blood
3. Ear, mastoid, sinus swabs
4. Eye swabs
5. Genital swabs
6. Skin, hair, nail scrapings or clippings
7. Spinal fluid
8. Sputum
9. Stool
10. Surgical biopsies
11. Throat and nasopharynx swabs
12. Transudates and exudates
13. Urine
14. Wound and ulcer swabs
Transudate versus exudate:
Transudates are characterized by pleural fluid/serum total protein ratio of 0.5 or less
and a pleural fluid/serum LDH ratio of 0.67 or less.
Exudates are characterized by a pleural fluid/serum total protein ratio of more than 0.5
and a pleural fluid/serum LDH ratio of more than 0.67.

Normal Flora
 Normal flora (also called indigenous flora, resident flora), are micro-organisms that
normally inhabit skin and mucus membranes.
 They normally do not cause disease in the host; they can do so if they gain entry to
a different area of the body.
 These organisms also play a role in protecting the host from pathogens. The Normal
Flora in man includes the following.

Body site Organisms GI Tract*

 Anaerobic bacilli (e.g., Bacteriodes & Clostridium spp.)
 Anaerobic cocci
 Gram-Negative enteric bacilli
 Enterococci
 S. aureus
 Yeast (Candida spp.)

Gastro Urinary - GU Tract

 Lactobacilli
 Anaerobic bacilli (e.g., Bacteriodes & Clostridium spp.)
 Anaerobic cocci
 Staphylococci (S. epidermidis & S. aureus)
 Enterococci
 Diphtheroids (Corynebacterium spp.)
 Streptococci
 Gram-Negative enteric bacilli (Acinetobacter)
 Yeast (Candida spp.)

Respiratory tract
 Staphylococci (S. epidermidis & S. aureus)
 Streptococci (e.g., viridans & pneumococci)
 Enterococci
 Diphtheroids (Corynebacterium spp.)
 Haemophilus
 Neisseria (N. meningitidis and Neisseria spp.)
 Gram-Negative bacilli (e.g., enterics and nonfermenters)
 Anaerobes
 Spirochetes
 Yeast (Candida spp.)
Skin
 Staphylococci (S. epidermidis & S. aureus)
 Micrococci
 Streptococci (nonhemolytic)
 Enterococci
 Diphtheroids (Corynebacterium spp.)
 Gram-Negative bacilli (e.g., enterics and nonfermenters)
 Anaerobes
 Yeast and fungi

*The large intestine may have up to 1011 organisms/gram of feces and most are
anaerobes.

Introduction to Infection
 Some microorganisms inhabit body normally and do not cause disease in their normal
site; these are known as normal flora
Pathogenic Microorganism: If microorganism causes disease to its host
 Bacteria enters the host via three (3) different pathways:
a) Inhalation
b) Ingestion
c) Direct Contact
 When bacteria enters the host, it will multiply in a reservoir (a comfortable place for
bacteria)
 Bacteria will multiply until there is no room in the reservoir, at this time reservoir will
burst and infection will occur

 There are three (3) modes of infection:

1) Direct - Sexual transmission,blood transfusion, etc.
2) Indirect -Through air, sharing glasses, etc.
3) Vector -By various insect bites (e.g. mosquito or fly)
 Some organism produce toxins which make them more virulent; the toxin
produces the disease; toxins that are destroyed by heat are called
thermolabile 

Stages of Infection - occurs in several stages:

1. Infection: Short period of time when contact was made with the microorganism
2. Invasion: Period of time when microorganism starts to grow and multiply
3. Prodromal: Period of time when the first symptoms are present
4. Acute: Period of time when all the symptoms are present
5. Decline Period: Host starts feeling better but is not yet well
6. Convalescence: Host is not sick but the body is still weak
7. Relapse: Re-occurrence of infection and usually worse than the first infection.
Results when host does not get enough rest during convalescence period or
incomplete antibiotic doses (it should be taken in full)

BASIC CONCEPTS OF PROPER COLLECTION  

Each specimen must be recorded in the accession log similar to other samples received
by the lab. (Follow SOP). The technologist decides what the procedure should be and
the types of media to be used. After the media has been inoculated a smear is made on
a microscope slide. Within the specified time for cultural methods, the cultures are
examined and a hanging drop preparation is prepared for motility if necessary. Further,
diagnostic procedures are performed using biochemical and serological reactions to
determine the identity of the pathogens present.

1. Specimen must be material from the actual infection site. A wound must be collected
from the depths, a sputum sample must not be contaminated with saliva, clean-catch
urine must be collected after adequate cleansing of the genitalia, etc.

2. The optimal times for specimen collection must be established. In some infectious
diseases, a positive blood culture would be obtained during the first week of illness; a
feces or urine culture is usually positive during the second a third week of illness.
Because of the rapid growth of some bacteria, 24-hour collections are discouraged.
Instead, first morning sputum and urine samples usually contain the highest
concentration of infectious organisms since the patient is less active during the night.

3. A sufficient quantity of specimen must be obtained. In active bacterial infection, it is

easy to obtain sufficient quantities of pus or secretions; in cases of mild or chronic
infection it may be more difficult. A dry swab or scant secretions are often submitted to
the lab "in the hope that something will grow." Such samples should not be discarded
since even a poor sample is better than no sample at all; but a second sample is usually
requested.

4. The correct collection devices, specimen containers, and culture media must be
used. Sterile containers should be used for the collection of all specimens. Containers
should be constructed for easy collection. Wide mouth bottles are best for sputum and
urine samples. The lid should be tight-fitting to prevent leakage or contamination during
transport.

Swabs are commonly used to collect specimens for culture. Specimens should remain
in contact with the swab no longer than necessary; to prevent drying out of the
organism. Swabs should be placed into a semi-solid transport medium (i.e., Stuart or
Amies transport medium). Throat, nose and tonsil swabs should be placed into clear
transport media. All other swabs should be placed into activated charcoal transport
media. The charcoal absorbs any toxins secreted by fast growing organisms. If not
removed, the toxin can inhibit the growth of slower growing organisms. Good recovery
of most bacteria is possible up to 24 hours after collection if the right transport medium
is used. Keep the time delay between specimen collection and specimen processing to
a minimum. 

5. If possible, obtain cultures prior to the administration of antibiotics. This is particularly

true when isolating beta-hemolytic streptococci from throat specimens, Neisseria
gonorrhoea from urethral or cervical secretions, or Hemophilus influenza and N.
meningitidis in cerebrospinal fluid. Antibiotic therapy may cause a false negative result
when the specimen is cultured.

6. The culture container must be properly labelled. Minimum information includes

patient's full name, identification number, physician's name or office in case a
consultation or early reporting is required, the specimen source, the date and time. This
ensures the specimen is cultured within an acceptable time after collection.

7. Handle the specimen with extreme care. It can potentially infect you, other staff
members and patients. Use Standard Precautions whenever you are handling these
biological cultures. Refer to the safety requirements of biohazard materials learned
earlier in this course.

8. Correctly decontaminate all work areas and materials after testing is complete.