Biology 333 Lab

Final Exam Review Sheet

James Jung

Laboratory Exercise 1
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Laboratory Exercise 2
 Negative Staining: Some bacteria are colourless and transparent in water. The negative stain creates a
dark background of suspended carbon particles on which the transparent organisms are easily visible.
The bacteria themselves do not take up the stain. It is useful for viewing bacteria which are difficult to
stain (such as spirochetes).
- Use India ink (nigrosine)

 The Acid-Fast Stain:

- Acid-fast bacteria contain large quantities of wax-like lipoidal material (mycolic acid) in their cell walls,
therefore most dyes cannot stain the cell wall.
- Carbolfuchsin is used to stain the cell wall. (Bacteria appear as red)
- There are two methods:
o Ziehl-Neelsen: uses heat to help the carbolfuchsin penetrate the cell wall
o Kinyoun: method uses alcohol.
 Mycobacterium species are Acid-Fast (Appear ad red) ex. Mycobacterium tuberculosis,
mycobacterium leprae, Mycobacterium Phlei
 Human Blood Cells
o Granulocytes
a) Neutrophils: Look for multilobed nucleus containing 1-5 lobes and fine granules
b) Eosinophils: present during inflammation, allergic response, and parasites are present. Look
for a bilobed nucleus with very large eosinophilic granules that stain orange-red

c) Basophils: play a role in inflammation and allergic responses. Look for: a bilobed nucleus
with large basophilic granules that stain violet-blue.
o Agranulocytes
a) Lymphocytes: are the second most numerous type of WBC. Look for: varying sized cells
with a large round, dark-staining nucleus, a light blue staining cytoplasm.

b) Monocytes: are the largest WBC that act as phagocytes. Look for: a large-kidney-shaped
nucleus, no granules, and often vacuoles in the cytoplasm.
Laboratory Exercise 3
- Hemolysis: is an important cultural attribute which involves the break down of blood cells on a blood
agar media. There are three different types of hemolysis
1. Beta-hemolysis: is the complete destruction of blood cells surrounding the bacterial colony. This
creates clear zones around the colonies.
2. Alpha-hemolysis: is a partial break down of the blood agar which results in a green “halo”
surrounding the colonies
3. Gamma-hemolysis: describes a plate where not blood cell destruction has taken place. There are no
clear zones around the colonies.
 Bacteria of the Skin and Respiratory Tract
Gram-Positive Cocci: Includes Micrococcus and Staphylococcus (Larger than Staphylococcus)
 Micrococcus: singles, pairs, irregular clusters, or tetrads.
Catalase: Positive. Oxidase: Positive
a) Micrococcus luteus: Cocci usually occur in tetrads and clusters. Colonies are smooth, golden
yellow and convex on nutrient agar (NA)
b) Micrococcus lylae: Cells arranged predominantly in tetrads. The colonies are non-pigmented or
cream-white, circular, convex and smooth on NA
c) Micrococcus roseus: Cells are in pairs and tetrads. Colonies are pink or red.
d) Micrococcus kristinae: Cells occur in tetrads and large clusters. Colonies are pale orange to
pale cream.
  Staphylococcus: is a part of the indigenous flora of the human body. Gram-positive,
nonmotile, aerobic, and facultatively anaerobic bacteria. It means ‘bunch of grapes’
(Smaller than Micrococcus)
Catalase: Positive, Oxidase: Negative)
Description Cellular Colony Hemolysis
Arrangement Characteristics
Staphylococcus Wound infection, Irregular clusters Golden yellow, Beta
aureus skin infections (acne circular, raised,
pimples), abscesses smooth
in bones and food
poisoning
Staphylococcus -opportunistic -pairs, tetrads, -Gray-white, large, Gamma
epidermis pathogen clusters circular mucoid,
glistening
Staphylococcus Common types of Paris and singles Large, circular, Gamma
saprophyticus urinary tract convex
infections include
cystitis (bladder),
urethritis (요도염), and
pyelonephritis.
Staphylococcus Second most Pairs and singles Large, circular Beta
haemolyticus frequently
encountered species
in clinical infections
such as septicemia
(페혈증), peritonitis
(복막염), urinary tract
infections and
wounds

Staphylococcus Aureus (Beta)

  Streptococcus Species: Gram-positive, microaerophilic or facultative anaerobic bacteria.
Some species are major causes of bacterial pneumonia (페렴) and sore throats. The
colonies of Streptococcus are much smaller than Staphylococcus.
Catalase: Negative, Oxidase: NA
Description Cellular Colony Hemolysis
Arrangement Characteristics
Streptococcus Respiratory pathogen Alpha
Pneumoniae Middle ear infection,
Endocarditis (심내막염: it is the
infection of the inner layer of heart,
endocardium) and meningitis
(뇌수막: the infection of inner layer that
covers brain)
-Sensitive to the chemical
optochin
Streptococcus -Causes “strep throat”, wound Pairs and long Beta
Pyogenes infection, skin infections, chain
puerperal fever, “flesh-eating
disease”.
-“pus-producing”
Streptococcus -inhabits the mucosa of the cheek Pairs and Alpha
mitis -isolated from teeth, sinuses, and chain
heart

Streptococcus -inhabits the tooth surface of man White or gray Alpha or

mutans -major cause of tooth decay. Rod non-
hemolytic
Enterococcus -isolated from human faeces Smooth, cream Gamma
faecalis -pathogenic agent in urinary tract or white
infections and endocarditis

1.
Streptococcus Pneumoniae (Alpha) Streptococcus Pyogenes (Beta)
- Causes endocarditis, meningitis - Causes “strep throat, skin infection
 Streptococcus Mitis (Alpha) Streptococcus mutans (Alpha)
- Isolated from teeth - Cause decay of teeth

o Gram-negative cocci
 Neisseria species: inhabit the mucous membranes of warm-blooded animals. Normal
flora of the upper-respiratory tract include N. mucosa, N. sublfava and N.sicca.
Catalase: Positive, Oxidase: Positive
Description Cellular Colony Hemolysis
Arrangement Characteristics
Neisseria -can be isolated from urethral and Diplococci Grey-white, Gamma
gonorrhoeae endocervical discharge with flattened convex
-It causes the sexually transmitted adjacent
disease gonorrhea edges
Neisseria -isolated from the nasopharynx Diplococci Gray or creamy Gamma
meningitidis -major cause of meningitis with flattened white
adjacent Round, smooth,
edges, some glistening and
encapsulated translucent
Moraxella -the normal flora of the upper Singles and Convex, almost Gamma
catarrhalis respiratory tract diplococci flat on aging
-infections of middle ear and
maxillary sinuses
-Cause meningitis and
endocarditis
 Neisseria mucosa Neisseria gonorrhoeae (Urethral pus)
Moraxella catarrhalis

- Gram negative
- Diplococci

o Gram-positive rods
Description Cellular Colony Hemolysis
Arrangement Characteristics
Lactobacillus -Large, gram-positive, -clusters -Gram positive
fermentative rods. -Chains and
-important in protecting the vagina pairs
from colonization and infection by -rods
pathogenic bacteria
-As cells from the vagina
epithelium are sloughed off, the
glycogen is released and the
lactobacilli ferment the glycogen
producing lactic acid. This lowers
pH which protects the vagina from
colonization and infection by
pathogenic microbes.
 Gardnerella -Gram-negative to Gram-variable, Small, round, Beta on
vaginalis pleomorphic, non-motile rod. smooth, and Vaginalis
-Major cause of bacterial vaginosis opaque agar (V-
agar)

Lactobacillus (Gram stain of vaginal smear) Gardnerella vaginalis

o Vaginal Infection
Description Cellular Colony Hemolysis
Arrangement Characteristics
Bacterial -It results from a decrease in the
Vaginosis lactobacilli normal flora, which
causes an increase in pH.
-The presence of “clue cells” in a
vaginal smear is one of the signs
of bacteria vaginosis. Clue cells
are exfoliated vaginal epithelial
cells that are covered with
bacteria.
- Vaginosis can lead to urinary
tract infections (UTI), pelvic
inflammatory disease (PID).
Candidal -Yeast infection Gram + Purple
Vaginitis -30-35% of vaginal infections, is
caused by fungal or yeast
colonization of the vagina.
- normal flora of the vagina
 - Opportunistic pathogen

Trichomonas -STD
Vaginitis -only 5-10% of vaginal infections
-is caused by the protozoan
Trichomonas vaginalis.
- It is a pear-shaped flagellated
protozoan.

1.
Bacterial Vaginosis Candidal vaginitis

Trichomonas vaginitis
Laboratory Exercise 4
 The Mannitol Salt Agar (MS): is selective and differential for Staphylococcus species. MS agar is
selective for Staphylococcus because most other bacteria are inhibited by the high salt concentration.
MS agar is differential because the media indicates the difference between a mannitol fermentor (yellow
plate) and a mannitol non-fermentor (pink plate). Only Staphylococcus epidermis is not fermentor.

 If the bacteria is alpha hemolysis: place an optochin (P)

 If the isolate is gamma hemolysis: place an SXT disk on plate

Staphylococcus epidermidis No, non-fermentor (pink)

Staphylococcus saprophyticus Yes, fermentor (yellow)

Laboratory Exercise 5

 Catalase test: to distinguishes between Staphylococcus and Micrococcus (Catalase Positive) and
Streptococcus (catalase negative). Neisseria and Moraxella are catalase positive. Catalase in an enzyme
found in most aerobic organisms which breaks down hydrogen perioxide to water and oxygen. A
positive test will be immediately indicated by bubbling O2.
 Oxidative test: to distinguishes Staphylococcus and Micrococcus. Micrococcus produce the enzyme
cytochrome oxidase whereas the majority of Staphylococcus species do not. Neisseria and Moraxella are
also oxidase positive. The purple/blue colored oxidase reagent is a redox dye that is oxidized by
cytochrome oxidase resulting in a deeper blue/black color only where the colonies are placed.
 API Staph-trac: is an identification system that provides a biochemical identification of Staphylococcus
species. The test involves inoculating a