MICROBIOLOGY BY:

Mzna Al-Muhaisen
mznmuhaisen@hotmail.com
 The main purpose of
microbiology laboratory is to
assist in the diagnosis of
infectious diseases by detecting
the bacteria that cause the
infection and identifying the
proper antibiotic therapy.

Specimens:
Urine, Stool, Swabs (nasal, eye, ear, wound or pus), Sputum, Tracheal
aspiration, Body fluids (synovial, peritoneal, pleural, ascetic or blood),
CSF, Semen

CONTAINERS

Sterile Culture and Transport Sterile Universal Container

Swabs.
 Ident ification of Microorganisms
Bacteriologic studies attempt to identify the specific organism causing an infection. This organism may be specific to one disease, such as
Mycobacterium tuberculosis for tuberculosis (TB), or it may cause a variety of infections, such as those associated with Staphylococcus aureus.

Cultures
The first step in processing clinical specimens is to provide an environment (media) on which the organisms will grow to numbers sufficient for
identification and other testing, such as antibiotic sensitivity procedures.
The source of the culture and the type of organism suspected will dictate the type of media on which the bacterial specimen is placed. Growth of
the organisms on the media is enhanced by using an incubator. A sufficient number of organisms must be grown to make possible morphological
studies and staining, the first steps in organism identification.

Culture Media
A variety of solid and liquid media are available for meeting the nutritive needs of bacteria and for growing them in numbers that allow for their
identification. Appropriate media must be chosen for inoculating and streaking plates for isolation of bacterial colonies.

Blood Chocolate
Mannitol
MacConkey
agar Agar Salt Agar XLD CLED
agar Enriched Selective (MSA) Selective
and and Enriched and Selective Differential
differential differential differential
used to isolate used to isolate used to isolate
bacteria from bacteria from bacteria from used in isolating
used to isolate
sputum, TA, throat sputum, TA, throat sputum,TA,,throat used for MRSA bacteria from stool and enumerating
\eye\ear\wound \eye\ear\wound \eye\ear\wound screening. sample. bacteria in urine.
swabs, body :luids, swabs, body :luids, swabs, body :luids,
CSF and pus. CSF, pus and stool. CSF and pus.

Procedure
1. Wash hands and don gloves.
2. Assemble necessary equipment and supplies. Use a biosafety
Cabinet class II
3. If reusable metal loops and needles are being used, turn on
incinerator and check that it has reached the optimum level of
heat.
4. Select the proper agar plate (Petri dish) and use the marker
to label it with appropriate identifying information.
5. Introduce the sterile swab or sterilized or disposable metal
loop into the sample.
6. Taking care not to touch any other surfaces with the swab or
metal loop, lift the lid from a sterile agar plate slightly and
Biological safety cabinet for working
inoculate a fourth of the plate by rolling the swab over the with contagious organisms. Class II biological Safety Cabinet
surface, taking care not to tear the agar.
7. Streak the remainder of the agar surface by touching the sterile loop to the first quadrant and streaking
all the way to the opposite side of the second quadrant, repeating the process 7 or 8 times. This serves to
isolate colonies of bacteria and to allow multiple strains of organisms to be separated.
8. Touch the loop to the second quadrant and streak all the way across the third quadrant, again repeating
the process 7 or 8 times
9. Streak the fourth quadrants to produce isolation of bacterial colonies in much the same manner as was
done for the second and third quadrants. Sterilize the inoculating loop or needle by using the incinerator
for several seconds/ dispose of the swab in a biohazard container.
10. Replace the lid on the inoculated agar plate and place it upside down in an incubator set at 37°C.

Incubator
Streaking method for urine sample

BACTEC: a fully automated system to culture and detect

growth of microorganisms in blood specimens.
 Morphology of the bacterial colony and cultural characteristics

MacConkey Agar - Escherichia coli MacConkey Agar - Salmonella enteritidis Chocolate Agar -
Note: Red colonies and red Note: Growth, but no fermentation of lactose. Colorless Staphylococcus aureus
precipitate due to acid production as colonies, medium is slightly yellow due to the increased
a result of lactose fermentation. pH resulting from bacterial digestion of peptone in the
medium.

Chocolate Agar - Sheep Blood Agar -

Neisseria gonorrhoeae Streptococcus pneumoniae,
Sheep Blood Agar -
Note: small colonies that alpha hemolytic
Streptococcus pyogenes, beta
appear transparent on
hemolytic
close examination.
 Sheep Blood Agar - E. coli,
gamma (non) hemolytic

Lactose fermenting (Yellow

colonies) and Lactose Non
fermenting colonies in CLED

Biochemical tests

Catalase Test

This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies
hydrogen peroxide by breaking it down into water and oxygen gas.

The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The
Staphylococcus spp. and the Micrococcus spp. are catalase positive. The Streptococcus and Enterococcus
spp. are catalase negative.

Oxidase Test

This test is used to identify microorganisms containing the enzyme cytochrome oxidase. It is commonly
used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.
A dark purple is considered a positive result.


Coagulase test

Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-positive, catalase
positive species to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor
of S. aureus. The formation of clot around an infection caused by this bacteria likely protects it from
phagocytosis. This test differentiates Staphylococcus aureus from other coagulase negative
Staphylococcus species.

 Staining reaction
Preparation of Smears
Principles
In some cases, exudates (discharge as from a wound) or body fluids such as cerebrospinal fluid (CSF) can be stained directly to gain critical
information regarding the type of bacteria that may be present. This is often attempted in cases where a quick presumptive identification is crucial
for the patient’s health, such as with organisms in CSF. Most slides for staining, however, are obtained from cultures. If mixed cultures of more
than one type of bacteria (as observed on an agar plate) are present, separate testing must be done on each type of bacteria. Bacterial smears for
staining must be heat fixed prior to initiating the staining process to prevent the organisms from washing off the slide during the staining
procedure.













Performing a Gram Stain
Principles
The most frequently performed stain in the microbiology department is the Gram stain. Staining of bacteria is necessary as it is virtually impossible
to see and describe the morphology of the organisms unless they are stained. Bacteria are small and
have little natural color for most species, so the Gram stain provides for differentiation as to the shape of
the organisms as well as providing further aid in identification by labeling the organisms as either Gram-
positive or Gram-negative. Differences in the cell walls of bacteria are based on their chemical
constituents, which in turn accounts for the staining difference. Gram-negative cell walls contain more
lipids (fats), polysaccharides, and lipoprotein complexes (including amino acids) than do those of the
walls of a Gram-positive organism. The cell walls of both Gram-positive and Gram-negative organisms
will absorb crystal violet, but Gram-negative organisms will lose the crystal violet in the decolorizing step,
and will then absorb the safranin, which imparts a pink to orange color to them. The Gram-positive
organisms will not lose their crystal violet during decolorizing and will appear purple in color.

The heat-fixed smear is placed on a staining rack and is flooded with crystal violet (the primary stain).
After 30 seconds to a minute, the slide is gently washed under a stream of water and returned to the
staining rack. Gram’s iodine is then poured over the smear. After 30 seconds to a minute, the slide is
again gently washed under a stream of water. The slide is then held at a 30- to 40-degree angle and
quickly rinsed with an acetone-alcohol mixture. This will decolorize the organism if it is a Gram-negative
species of bacteria. The decolorizing process only takes a few seconds. All color should run from a slide
containing predominantly Gram-negative organisms. Finally, a counterstain (safranin) is poured over the
slide. Gram-negative organisms absorb and retain the counterstain.















Performing an Acid-fast stain
Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These
acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia.
The reagents used are Ziehl–Neelsen carbol fuchsin, acid alcohol, and methylene blue. Acid-fast bacilli will be bright red after
staining.





Mycobacterium tuberculosis visualization using

the Ziehl–Neelsen stain.



 Antibiotic Susceptibility Test ing
The susceptibility test detects the type and amount of antibiotic or chemotherapeutic agent required to inhibit bacteria growth. Often, culture and
susceptibility tests are ordered together. Susceptibility studies also may be indicated when an established regimen or treatment is to be altered.

A common and useful test for evaluating antibiotic susceptibility is the disk diffusion method. A set of antibiotic-impregnated disks on agar is
inoculated with a culture derived from the specific bacteria being tested. After a suitable period of incubation, the degree of bacterial growth
within the different antibiotic zones on the disks is determined and measured. Growth zone diameters, measured in millimeters, are correlated to
the minimum inhibitory concentration (MIC) to determine whether the organism is truly susceptible to the antibiotic. Nowadays, many automated
machine are used for susceptibility tests.

MicroScan AND Phoenix: Automated machines for microorganism identification and susceptibility testing.