Microbiology II Final Lab Exam Study Guide

The introduction and observation sections of Genus Streptococcus
o The genus Streptococci contains a variety of species that will cause
disease in humans. The group contains Gram positive cocci that occur in
pairs or chains. They are a non-motile, non spore-forming group of
bacteria that are negative for catalase production and typically attack
media containing red blood cells. Most members are facultative
anaerobes, although some members are strict anaerobes. They are
fastidious in their nutritional demands and require a variety of substances
that must be added to the medium in order to support growth. There have
been several attempts to devise a classification scheme for the genus, but
the method that is widely accepted was proposed in the early 1930s by
Rebecca Lancefield. It is a serological classification based on serologic
differences in an extractable carbohydrate in the cell walls of members of
this genus. The five serological groups Lancefield identified are called A,
B, C, D, and E. Additional groups have since been identified and the
number is presently at >18. Group A is the most pathogenic group
containing Streptococcus pyogenes, which is the causal agent of strep
throat, as well as, erysipelas, impetigo, scarlet and rheumatic fever,
glomerulonephritis, and necrotizing fasciaitis. Streptococcus pneumoniae
is also a member of Group A. It is an encapsulated bacterium that causes
lobar pneumonia, but also causes otitis media, sinusitis, meningitis,
bacteremia, and endocarditis. The capsule has been shown to be an
important virulence factor. Serologic identification of S. pneumoniae is
enhanced because of the variety of capsular types. At present, there are
more than 80 different types because of the differences in polysaccharide
components in the capsule. Certain serotypes are epidemiologically
important, whereas others are rarely associated with disease. Virulent
strains produce moist, mucoid colonies because of the capsule, whereas
nonencapsulated strains produce a translucent colony that is initially
convex. With time the colony develops a central depression, a useful
identification characteristic. Microscopically, pneumococci are lancetshaped coccoidal cells that appear as Gram-positive diplococci but
sometimes develop into short, tight chains or singly.Group B contains the
species Streptococcus agalactiae, which causes neonatal sepsis,
pneumonia, and meningitis.Group D houses Streptococcus faecalis, which
causes urinary tract infections.Streptococcus species are commonly found
in the naso- and oropharyngeal cavity. Since there are many other bacteria
found there also, it is important to have methods to distinguish between
them.
o Important characteristics of this genus are:
 Cell and colony morphology
 Hemolysis on blood agar
 Bacitracin susceptibility
 Growth in 6.5% NaCl


Test
Hemolytic type
Bacitracin
susceptibility

Growth in methylene blue milk
Esculin hydrolysis
S. pyogenes
Beta
S. agalactiae
Beta
+
-
S. faecalis
Alpha or
Gamma
-
S. pneumoniae
Alpha
+
The introduction and observation sections of Family Enterobacteriaceae
o The enterics are an important group of Gram negative, non-sporeforming
bacilli. They inhabit the intestinal tract of man and belong to the family
Enterobacteriaceae. This is a large group of bacteria and is composed of a
significant number of important species. Members of this family are often
referred to as coliforms. More frequently, however, the term is used to
describe only the fermentative inhabitants of the intestinal tract such as
Escherichia coli, Klebsiella pneumoniae, and Enterobacter aerogenes.
This group is known to contain species that cause a variety of infectious
diseases, including typhoid fever, bacillary dysentery, diarrheal disease,
bacteremia, meningitis, urinary tract infections, respiratory infections, and
wound infections. In addition to the many pathogens in this family there
are also a number of commensals in the group that can produce disease in
the compromised patient. Patients with underlying disease or those who
are immunosuppressed are particularly susceptible to the enterics. This
family is responsible for 50% of nosocomial infections, most frequently
caused by E. coli, Klebsiella, Enterobacter, Proteus, Providencia, and
Serratia marcescens. The microbes of this family are distributed
worldwide. They are found in soil, water, fruits, vegetables, grains,
flowering plants and trees, and animals from worms and insects to
humans. Escherichia coli is the most common facultative inhabitant of the
intestine measuring 107-108 per gram of feces.Because the family contains
so many important disease-producing genera, it is the most frequently
encountered group of bacteria in the clinical laboratory. Although they are
normal inhabitants of the intestinal tract, they may be found in other
clinical specimens such as blood and sputum. Although major
characteristics separate individual species, they have many characteristics
in common such as the ability to produce catalase of ferment glucose.
Most are motile. Most laboratories have definite steps that are followed to
identify an isolate. These steps are usually directed at showing Salmonella
or Shigella to be the cause of the patient’s disease. As suggested,
however, many other genera in the family may be involved. Perhaps the
most important characteristics used to separate Salmonella and Shigella
from the others is lactose utilization. These genera give a negative
reaction while other enterics usually give a positive reaction. Escherichia
and Enterobacter give a strong positive reaction, but Proteus is negative
and is sometimes confused with the non-lactose fermenters.
o Clinical specimens that are to be analyzed for enterics are usually taken
through a series of procedures intended to aid identification as shown in
the following table.
Steps in the Identification of Enterics
1. Inoculate enrichment broth
2. Inoculate differential/selective media
3. Inoculate TSI agar
4. Determine biochemical reactions
5. Determine serological properties
 The specific characteristics that follow should be used in the identification
of Enterobacteriaceae.
 Cell and colony morphology
 Motility
 Fermentative metabolism
 Glucose utilization
 Ornithine decarboxylation
 Oxidase reaction
 Nitrate reduction
 End-product identification
 Unfortunately, there is no single morphological or biochemical
characteristic that can be used to distinguish individual members.
 Differential and selective plating media are useful for distinguishing
particular microorganisms from others. Selective media allow growth of
only one group while differential media allow growth of several groups
but particular properties of the organism can be distinguished from others.
Occasionally a single medium may have both selective and differential
properties.
 The clinical microbiologist may want additional information concerning
the biochemistry of an isolate. For that reason, there are numerous media
and reagents in self-contained testing systems that give biochemical
profiles of the microorganism in question. With the use of identification
charts, results provide the name of the isolate.
 The following table contains information that will be useful for
identification of the species under study.
Test
E. coli
S. typhi
S. dysenteriae
K. pneumoniae
TSI-slant
+
+
TSI-H2S
+/- (don’t
production
study, it’s
variable)
TSI-gas
+/- (don’t
+/- (don’t
+
production
study it’s
study it’s
variable)
variable)
Simmons
+
+
Citrate
Motility



+
+
-
-
Know the steps necessary to identify an unknown. What is the first step?
o First step in identifying an unknown
 Prepare a smear for staining. Perform a Gram stain and note the
reaction and morphology.
 From these determinations, decide what media and/or biochemical
tests will be necessary to continue your identification.
Introduction and observation sections of Staphyloslide
o The Staphyloslide Latex test is an agglutination test for the differentiation
of staphylococci which possess clumping factor and/or Protein A, usually
present with Staphylococcus aureus, from staphylococci that do not
possess these properties. Approximately 97% of human strains of S.
aureus possess bound coagulase. This is evident by coagulase testing that
detects clumping/clotting factor (bound coagulase) on the bacterial cell
surface. Protein A is found on the cell surface of about 95% of human
strains of S. aureus and has the ability to bind the Fc portion of
immunoglobulin G.The Staphyloslide Latex test consists of blue latex
particles coated with human fibrinogen and IgG. On mixing the latex
reagent with staphylococci colonies that possess Protein A or bound
coagulase, cross linking will occur causing an agglutination reaction. This
occurs most notably with S. aureus. If neither bound coagulase nor
Protein A are present, no agglutination will occur and the result will be
negative. This occurs most frequently with Staphylococcus epidermidis.
o PAY ATTENTION TO WHAT MAKES THEM POS AND NEG
o Positive Result: A positive result is obtained if agglutination or the blue
latex particles occurs within 20 sec in the test circle, with no agglutination
in the control circle. The result is positive when there is noticeable
clearing of the blue background in the test latex. This indicates the
presence of S. aureus.
o Negative Result: A negative result is obtained if no agglutination occurs
and a smooth suspension remains at 20 sec in the test circle. The result is
negative when there is no noticeable clearing of the blue background in
the test latex. *Reactions occurring after 20 sec should be ignored.
o Staph Aureus was positive
o Staph Epidermidis was negative
Introduction and observation sections of PYR Dry Slide
o The DrySlide is used for detecting hydrolysis of L-pyrrolidonyl-βnaphthylamide (PYR). This indicates the presence of the enzyme
pyrrolidonyl peptidase. This is a test for rapid presumptive identification
of A streptococci and group D enterococci. 98% of group A and 96% of
group D streptococci hydrolyze PYR. Nearly 100% of all other groups
and species of streptococci yield a negative PYR test result. While this
test may be used as a presumptive diagnostic aid for identifying these
organisms, additional biochemical and serological tests should be
performed for complete identification.


o Positive Result: PYR positive organisms change the color of the reaction
area from pale yellow to pink/fuchsia within 1 minute of adding the color
developer. This indicates the presence of either a Group A or Group D
streptococcus species.*Some streptococcal strains produce only a small
amount of enzyme, so the color change is weak. A weak positive reaction
can be enhanced by allowing the test organism to incubate 5 minutes
rather than 2 minutes at room temperature before adding the color
developer. (HE SAID PAY ATTENTION TO THIS PARAGRAPH)
o Negative Result: A negative result is obtained if no color change from
yellow occurs upon addition of the color developer within 1 minute.
o S. pyogenes was positive
o S. agalactiae was negative
Be able to tell me how you inoculated the enterotube, how long it was incubated,
and how you found out what organism you had.
o Inoculation of Enterotube by first twisting the wire, then withdrawing it
through all twelve compartments using a turning motion.
o Reinsert wire into Enterotube using a turning motion through all twelve
compartments, until the notch on the wire is aligned with the opening of
the tube. The tip of the wire should be visible in the citrate compartment.
Break the wire at the notch by bending.
o Punch holes with the broken off part of the wire through the foil covering
the air inlets of the last eight compartments (adonitol, lactose, arabinose,
sorbitol, Voges-Proskauer, dulcitol/PA, urea, and citrate) in order to
support aerobic growth in these compartments. Replace both caps.
o Incubate at 37oC for 24 hours with Enterotube lying on its flat surface.
o Indicate each positive test reaction by circling the number appearing
below the appropriate compartment on the Results Pad.
o Add the circled numbers in the bracketed section and enter the sum in the
space provided below the arrow.
o Locate the five-digit number in the CCIS and find the best answer in the
column entitled “ID Value”. NOTE: Should more than one organism be
listed, the confirmatory test required to correctly identify the organism
will be indicated in the CCIS.
Introduction and observations of the Genus Staphylococcus
o The genus Staphylococcus is one of the most important members of the
family Micrococcaceae, which also includes Streptococcus and
Micrococcus. The staphylococci are a medically significant genus in that
Staphylococcus aureus is the number one causative organism for skin
abscesses. Furthermore, species from this genus are known to cause
cellulitis, impetigo, bacteremia, endocarditis, brain abscesses,
osteomyelitis, urinary tract infection, toxic shock syndrome, scalded skin
syndrome, and food poisoning. The staphylococci are Gram-positive
cocci occurring singly, in pairs, and irregular clusters. They are
facultative anaerobes with both oxidative and fermentative metabolism.
Colonies are usually opaque and may be white or cream and sometimes
yellow to orange. They are usually catalase positive; nitrate is often
reduced to nitrite, and grows well at high osmotic pressures (20% NaCl).
There are specific methods, which are used to separate the staphylococci
from other Gram-positive cocci as well as methods that are used to
distinguish each species of the genus. Important characteristics that are
used to identify these microorganisms are listed below:
1. Cell and colony morphology
2. Growth on Mannitol Salt Agar
3. Pigment production
4. Hemolysis on Blood agar
5. Catalase production
6. DNAse production
7. Coagulase production
8. Novobiocin resistance
9. Fermentation of mannitol
Nitrate reduction
Hemolytic type
DNAse production
Novobiocin
resistance
S. aureus
+
Beta
+
-
S. epidermidis
Variable (don’t
study)
Gamma
-
S. saprophyticus
Gamma
+