1. Week 10W New This Week: Last week: Gram (+) Bacteria ID
Review Gram + and - tests
Last week:
Exp 62: ID of Human Staph. and Strep. Pathogens
Lab Two, Part 2: Blood and MSA, pg 429
Lab Two, Part 1: Coagulase Test, pg 429
Physical Factors
Exp 15: Temperature
Exp 16: pH
Exp 17/18: O2 Requirements
Exp 29: Catalase Test
Lab Two, pg 196
add H2O2
Gram staining practice
Week 10W 1

2. Syllabus Corrections Pg 3 Pg 8 Week 12: Assign. #7 due
Week 13: Assign. #6 due (not # 8)
Pg 8
Week 11: Prepare Assign. #7 (not #6)
Week 12: Quiz 9 covers definitions from Assign. #7 (not #6)
: Prepare Assign. #6 (not #8)

3. Exp 62: ID of Human Staph. And Strep. Pathogens: Blood and MSA Review
  Red blood cells on an agar plate are used to diagnose infection. On the left is a positive Staphylococcus infection, on the right a positive Streptococcus pyogenes culture.
Selective & Differential Media
a. Blood agar: for fastidious bact. (streptococcus, styphylococcus, etc.)
Hemolysin – destroy red blood cell
Alpha-hemolysis: incomplete hemolysis – green ring
Beta-hemolysis: complete hemolysis – clear zone
Gamma-hemolysis: absence of hemolysis
b. Mannitol salt agar: Selective - most bacteria cannot grow except
staphylococcus due to high salts
Differential- Mannitol-fermenting species of Staph
will change the phenol-red color. Other Staph will grow without color change.
An MSA plate with Micrococcus sp. (1), Staphylococcus epidermis (2) and S. aureus (3) colonies. 3

4. Exp 62: ID of Human Staph. And Strep. Pathogens: Coagulase Test
Coagulase reacts with the fibrinogen in plasma, and precipitates into fibrin cells clump together , look “lumpy”.
Test useful for differentiating Staphylococcus aureus from other Gram positive, catalase + cocci.
Any degree of clotting, from a loose clot suspended in the plasma to a solid, immovable clot is a positive result. Plasma which liquid remained is a negative result.
The coagulase test identifies whether an organism produces the exoenzyme coagulase (attached to bacteria’s cell wall), which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents.
Staphylococcus aureus produces coagulase while Staphylococcus epidermidis and Streptococcus salivarius do not.
Staphylococcus aureus is a common cause of food poisoning (through the production of an exotoxin) and is also the most frequent cause of boils. It is frequently found in nosocomial infections. Pathogenic Staphylococci characteristically produce coagulase enzymes that coagulate plasma. The production of coagulase is a characteristic indicator that a strain of Staphylococcus is S. aureus. In Public Health Laboratories the assay for coagulase activity is now usually performed using latex bead agglutination, however the test below is the classical assay for coagulase activity and a positive result produces a clearly visible veil of polymerized fibrin. 4

5. Exp 62: ID of Human Staph. And Strep. Pathogens: Coagulase Test
The coagulase test identifies whether an organism produces the exoenzyme coagulase (attached to bacteria’s cell wall), which causes the fibrin of blood plasma to clot. Organisms that produce catalase can form protective barriers of fibrin around themselves, making themselves highly resistant to phagocytosis, other immune responses, and some other antimicrobial agents.
The coagulase test identifies the presence of coagulase which is attached to the cell walls of the bacteria. Coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate to fibrin. This causes the cells to clump together which creates the “lumpy” look of a positive coagulase slide test.
The coagulase test is useful for differentiating potentially pathogenic Staphylococcus aureus from other Gram positive, catalase positive cocci.
Any degree of clotting during this time, from a loose clot suspended in the plasma to a solid, immovable clot is a positive result. Staphylococcus aureus produces coagulase while Staphylococcus epidermidis and Streptococcus salivarius does not.
Staphylococcus aureus is a common cause of food poisoning (through the production of an exotoxin) and is also the most frequent cause of boils. It is frequently found in nosocomial infections. Pathogenic Staphylococci characteristically produce coagulase enzymes that coagulate plasma. The production of coagulase is a characteristic indicator that a strain of Staphylococcus is S. aureus. In Public Health Laboratories the assay for coagulase activity is now usually performed using latex bead agglutination, however the test below is the classical assay for coagulase activity and a positive result produces a clearly visible veil of polymerized fibrin.
fibrin 5

6. Exp. 29: Catalase Test accumulation is toxic to cells.
Hydrogen peroxide (H2O2)
accumulation is toxic to cells.
Presence of bubbling or foaming
when adding H2O2 to a culture,
indicates that the hydrogen
peroxide is being broken down
by catalase, with the release of
oxygen gas.
Bubbling = pos. catalase
Absence of foaming/bubbling=
neg. catalase
(catalase is not present in cell)
. Observe for presence or absence of bubbling or foaming. Presence indicates that the hydrogen peroxide is being broken down (by catalase), with the release of oxygen gas (causes the bubbling) = positive catalase. The absence of foaming/bubbling indicates that catalase is not present= negative catalase
You may find colonies of Staphylococcus epidermidis difficult to distinguish from non-haemolytic Enterococci. However, Gram-stained Staphylococci may be seen as grape-like clusters of round cocci whereas non-haemolytic Enterococci usually form pairs of smaller, oval cocci.. A further distinguishing feature is provided by the catalase test in which oxygen is visibly liberated from H202 by Staphylococci, but not by Enterococci or streptococus 6

7. Results To ID Gram Positive Bacteria Staph. epidermidis (SE)
Notation
MICROORGANISM
Staph. epidermidis (SE)
Staph. aureus (SA)
B. subtilis (BS)
Strep. salivarius (SS)
M. luteus (ML)
Morphology
shape/ arrangement
cocci/ cluster
rods
cocci/ chain
TSA (cultural characteristics)
color, size, form, margin, elevation, texture
+/- growth ■
MSA
MF or NMF/ color
Blood Agar (cultural characteristics)
Blood Hemolysis
У α β
Catalase Activity
+/- catalase
Coagulase Actvity
+/- coagulase
Streptococcus salivarius is a species of spherical, Gram-positive bacteria which colonize the mouth and upper respiratory tract of humans a few hours after birth, making further exposure to the bacteria harmless. The bacteria is considered an opportunistic pathogen, rarely finding its way into the bloodstream, where it has been implicated in septicemia cases.
Streptococcus salivarius is the principal commensal bacterium of the oral cavity in humans. S. salivarius is a normal inhabitant of the upper respiratory tract. It may enter the blood stream by accident during dental work or when brushing the teeth. It is the first bacterium which colonizes the dental plaque, before being joined by numerous other species of various genera [6]. It therefore seems to be the pioneer in colonizing dental plaque, it creates favorable conditions so other species can begin to colonize. It is also a bacterium which plays the role of moderator, permitting the implantation of bacteria which are harmful to the health of the oral cavity.
Better knowledge of the molecular and physiologic factors which allow it to colonize dental plaque and to interact with other species will help in designing strategies for the prevention of cavities, especially in children [6]. Also, greater knowledge of this organism can help with research on mouth odor.
GO OVER GRAM POS TREE TO ID UNKNONW
GO OVER ALL TEST PROCEDURES 7

8. Biofilm build-up on tank walls—guaranteed to contaminate any fuel
Grand Prismatic Spring, Yellowstone National Park (thermophiles)
Bacteria Around Nuclear Reactors
PHYSICAL
FACTORS
Below Freezing
Salt Water Pockets in Antarctica
Seafloor bacteria on ocean-bottom rocks are more abundant and diverse than previously thought.
Bacteria live in the most diverse range of environments of any forms of life. There are bacteria that live in salt water below freezing in melted ice pockets in the Antarctic, in saturated salt solutions, in boiling hot springs, in acid waters at pH <2.0, around the core of atomic reactors, in the fuel tanks of airplanes, at the bottom of the ocean at >110oC and hundreds of feet below the surface of the ground. A basic principle of microbial growth is that every microbe has a PREFERRED ENVIRONMENT and it is a challenge to the investigator to tease out the unique set of conditions, called the OPTIMAL GROWTH CONDITIONS that suits each microbe best. It is estimated that we currently are able to cultivate only a few percent of the 100,000s of bacterial species. The reason for this is evolution. Every microbe has evolved (over 3.5 billion years) to fit into a UNIQUE ENVIRONMENT which gives it MAXIMUM SURVIVAL POTENTIAL. Everyone of a bacteria's 2 to 4,000 genes has been designed for the purpose of maximum survival in a unique environment, called its NICHE.
Grand Prismatic Spring, Yellowstone National Park, captures the essence of Yellowstone's world-renowned geysers and hot springs that are fed by the enormous heat from its active volcanic system. This 60 meter-wide, boiling hot spring has an array of vivid colors produced by thermophile bacteria that thrive in hot water.
Biofilm build-up on tank walls—guaranteed to contaminate any fuel 8

9. Exp 15: Physical Factors: Temperature
Temperature: Temperature primarily affects the enzymes of a microorganism: a rise in temperature increases enzyme activity and allows a faster growth rate, until key enzymes are denatured. The temperatures at which these events occur vary widely amongst microbes, which all have characteristic maximum, minimum and optimum temperatures for growth. Organisms which inhabit the human body as commensals and/or pathogens are mesophiles, and grow most rapidly within the range 20·C to 45·C, with growth optima between 35·C and 40·C.
Psychrophile
cold-loving
-5o - 20o C
Mesophile-
moderate temps. – will grow at body temps
20o - 45o C
most pathogens 37o C
Thermophile -
heat loving
35o C 9

10. Exp 16: Physical Factors: pH of the Extracellular Environment
Figure 4. Growth rate vs pH for three environmental classes of procaryotes. Most free-living bacteria grow over a pH range of about three units. Note the symmetry of the curves below and above the optimum pH for growth.
Table 8. Minimum, maximum and optimum pH for growth of certain procaryotes.
pH: Most bacteria have an optimum pH for growth in the range with limits somewhere between 5 and 9. Acidophilic bacteria can grow at a low pH, and such organisms are very important in Oral Microbiology as the causative agents of caries: lactobacilli and mutans streptococci produce acid as end products of metabolism of dietary sugars, and are able to survive and grow in the acidic conditions created (aciduric). 10

11. Exp 17/18: Physical Factors: Atmospheric Oxygen Requirements
Category
Oxygen Requirement
Production of Catalase
Obligate Aerobe
Requires oxygen and oxygen is not toxic to it.
Produces catalase.
Microaerophile
Requires oxygen at low levels and atmospheric levels of oxygen are toxic to it. Prefers oxygen at a concentration lower than 10%.
Produces catalase sometimes.
Facultative Anaerobe
Uses oxygen preferentially when oxygen is present, but can grow without oxygen (slower growth than with oxygen). Oxygen is not toxic to it.
Obligate anaerobe
Does not use oxygen and oxygen is toxic to it.
No catalase production.
Aerotolerant
Does not use oxygen, but oxygen is not toxic to it.
Oxygen is a universal component of cells and is always provided in large amounts by H2O. However, procaryotes display a wide range of responses to molecular oxygen O2.
Obligate aerobes require O2 for growth; they use O2 as a final electron acceptor in aerobic respiration. (eg Mycobacteria)
Obligate anaerobes (do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis. Do not produce catalase or superoxide dismutase. (eg Clostridium)
Facultative anaerobes are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O2 they switch to aerobic respiration. (Prefer resp w/O2 otherwise may resp w/nitrates, sulfates or just do ferment. Although they growth is slower.) (eg Streptococcus, staphylococcus, enterobacter)
Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O2. They live by fermentation alone whether or not O2 is present in their environment. Prod catalase and superoxide dismutase. (eg Lactobacillus)
Microaerophiles require O2 in limited qty. (egNesseria gonnorrhaea) Superoxide dismutase, catalase and peroxidase are enzymes that detoxify oxygen radicals which are inevitably generated by living systems in the presence of O2. The distribution of these enzymes in cells determines their ability to exist in the presence of O2.
How is distribution of growth affected by atmospheric oxygen ……..? 11

12. Week 10W SUC GLU LAC TABLES 1 & 2 Exp 15: Temperature
Media: 12 NUT slant
Culture: SA , ML, BST
Procedure: (refer to pg. 111)
Label each tube w/ org. and temp.
Inoculate with loop 4 slants w/each org.
Incubate 1 slant from each org. at either 4oC, 25oC, 37oC, and 55oC for 24 hrs.
TABLES 3 & 4
Exp 16: pH of the Extracellular Environ.
Media:
2 NUT pH 2
2 NUT pH 5
2 NUT pH 7
2 NUT pH 9
Culture: EC & SA
Procedure: (refer to pg. 116)
Label each tube w/
org. and pH.
Inoculate with ea. org in various pH tubes.
Incubate at 37oC for 24 hrs.
BST- Bacillus
stearothermophilus
CS- Clostridium
sporogenes
TABLES 5 & 6
Exp. 17/18: Atmospheric
Oxygen Requirements
Media:
3Thioglycollate broth
3 NUT broth
Culture: EC, ML & CS
Procedure: (refer to pg. 127)
Label ea. tube w/org & media.
Inoculate each org. in
thioglycollate and NUT broth.
Place thioglycollate tubes in
anaerobic GasPack chamber.
Incubate all at 37oC for 24 hrs.
Anaerobic Jar
Anaerobic conditions can be created by removing O2 from a container
Use a gas pack that generated H2
Hydrogen gas combine with O2 in jar to form H2O for a CO2 environment
Need Palladium catalyst to accelerate reaction
Fluid Thioglycollate- medium contains Na thioglycollate which binds O2 gas, thus acting as areducing compound. Also present is a redox potential indicator that produces a pink color in an oxidixed state (if O2 is in solution).
SUC
GLU
LAC
Week 10W
Gram staining practice 12