Final Set - Lab Final

Simple stain

This slide was created by adding bacteria then heat fixing. After heat fixing a single stain was added then rinsed with water. What type of stain is this?

Negative stain

This slide was created by adding a drop of an acidic dye to a slide then adding bacteria with a loop. This mixture was then spread across the slide using a second slide. Once this slide dries it is ready for viewing. What type of stain is this?

Differential stain

This slide was created using two different stains, which allows for the detection of differences between organisms or even parts of the same organism. What type of stain is this?

Basic stain

This slide was created using a cationic or positively charged stain. The use of the positively charged stain allows it to bind the negatively charge cell wall, thus staining the cell. What type of stain is this?

Acidic stain

This slide was created using a negatively charged stain. Because the stain carries a negative charge it is repelled away from the negatively charged cell walls. What type of stain is this?

Gram stain

This slide was created using two different stains: crystal violet and safranin. This technique is used to differentiate between gram positive and gram negative bacteria. What type of stain is this?

Capsule stain

This stain was made using two different stains. This stain is used to detect cells capable of producing biofilm. What type of stain is this?

Endospore stain

This is a differential stain used to detect the presence and location of spores in bacterial cells. What type of stain is this?

Starch hydrolysis

The above test is used to determine what?

Starch agar

What type of agar is used in this test?

Positive

Would this be considered a positive or negative starch hydrolysis test?

Flagella stain

This stain is used to visualize flagella. In order to do so a mordant is used to encrust flagella with stain to a visible thickness. what type of stain is this?

Sugar fermentation test

The above test is used to determine sugar fermentation. What test is this?

Glucose, lactose and sucrose

The above test is used to determine sugar fermentation. It has the ability to test for the fermentation of three different sugars. Name these sugars:

gas production, Durham Tube

The above test has a glass vial inside the tube. What does this vial test for? What is it called?

Phenol Red

What indicator is used in the above sugar fermentation test?

The first yellow tube (A/-)

Which on of these tubes would be positive for sugar fermentation with no gas produced when running a sugar fermentation test?

The red tube furthest to the right (-/-)

Which one of these tubes would indicate no fermentation when running a sugar fermentation test?

The pink tube (K)

Which one of these tubes would indicate the organism is able to break down sugars into peptone when running a sugar fermentation test?

The second yellow tube (A/G)

Which one of these tubes would indicate that the organism is able to ferment sugar and also has a gas by product when running a starch hydrolysis test?

Motility

The above test is used to determine what?

The first and last tubes

Which of the above tube(s) would be considered positive for motility?

Motility test medium

What type of agar is used in the above test?

Catalase test

The above image is of what test?

The sample of the right

Which one of these would be considered a positive result for a catalase test?

H2O2, hydrogen peroxide

What reagent is used in the above test?

The organism grown on the plate produces the enzyme catalase

What does this test determine?

Oxidase test

The above image shows what test?

Positive

Would this be a positive or negative oxidase test?

Chromogenic reducing agent

What reagent is used in the above test?

Nitrate reduction test

The above test is used to determine whether an organism reduces nitrate to nitrite. What is this test called?

Tube 2

The above tubes have been incubated for 24 hours, after incubating all four tubes remained yellow with no bubble formation. Thus, 8 drops of reagent A and B were added to tubes 1 and 2. Which tube indicates a positive result, 1 or 2?

Tube 3

After adding reagent A and B to tubes 3 and 4 no color change was seen, both tubes remained yellow. Zinc power was then added, which tube would be considered a positive result tube 3 or 4?

Urease test

The above test is used to differentiate organisms on their ability to hydrolyze urea using the enzyme urease. What is this test called?

Phenol red

What is the indicator used in the urease test?

The pink tube (+)

Which on of the above tubes is positive for urea hydrolysis?

Both the yellow and orange tubes (-)

Which one of these tubes is negative for urease hydrolysis?

Antibiotic resistance and susceptibility

The above Kirby-Bauer test is used to determine what?

Mueller Hinton

What medium is used for the above Kirby-Bauer test?

The zone of inhibition

In order to test for susceptibility in the Kirby-Bauer test, what do you measure?

Staph aureus

The above image shows three different species of staph grown on MSA. What species is most likely growing in section 1?

Mannitol fermentation and the reduction of the pH

What caused section 1 of this plate to turn yellow?

Gram-negative organisms

Does the above MSA plate select for gram-positive of gram-negative organisms?

Staphylococcus

What organism does the above medium favor the growth of?

Coagulase test

The above test is used to determine whether an organism has the ability to clot. What is this test called?

The bottom tube

Which one of these tubes would be considered a positive result when running a coagulase test?

To differentiate S. aureus from other gram-positive cocci

Why would you run a coagulase test?

CAMP

The above image is an example of what test?

It can be used to identify Streptococcus agalactiae. Though not strongly beta-hemolytic on its own, it presents with a wedge-shape in the presence of Staphylococcus aureus.

What does the CAMP determine?

Beta hemolysis

The above organism is growing on blood agar, what type of hemolysis is seen?

Alpha hemolysis

The above organism is growing on blood agar, what type of hemolysis is seen?

Gamma hemolysis

The above organism is growing on blood agar, what type of hemolysis is seen?

Bacitracin

The above test is used to test susceptibility to what antibiotic?

Beta, streptococci, beta

Both of the organisms on the above plate display ________________ hemolysis. The bacitracin susceptibility test is often used to differentiate group A __________________ (strep pyogenes) from other _________________ hemolytic streptococci. Group A strepto

Blood agar

The above bacitracin susceptibility test is run on what medium?

Bile esculin

The above test is grown on what medium?

Positive, any darkening of the medium is considered positive

On the above bile esculin plate would this be considered a positive or negative result?

Enterococci, Streptococcus bovis

Bile esculin is used for presumptive identification of __________________ and members of the _______________________ ___________________ group.

MacConkey agar

The above plate is yellow before inoculation. What type of agar is this?

Neutral red

What is the indicator in MacConkey agar?

Gram-negative

MacConkey agar inhibits the growth of gram-negative or gram-positive organisms?

Ferment lactose

MacConkey agar is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to _______________________ ____________________ (turns the medium pink).

Coliform

The organism growing on the right side of this MAC plate is a probable _________________ based on the color change and bile precipitate.

EMB, eosin methylene blue

The above plate is inoculates with a gram-negative organism. The organism displays good growth as well as a metallic sheen hue, what is the medium?

Gram-negative

EMB selects for gram-negative of gram-positive organisms?

Coliform

The above organism seen growing on EMB is a probable __________________ based on the amount of growth as well as the metallic sheen color.

Sulfer production, indol, moltility

SIM medium can be used to run three tests. Name these tests:

black precipitate

What would a SIM tube positive for hydrogen sulfide production look like?

Red band on top of medium

What would a SIM tube positive for indole look like?

Entamoeba histolytica

The above organism is a protozoan that belongs to the phylum Sarcomastigophora and the subphyllum Sarcodina. Name this organism

Giardia lamblia

The above image is a protozoan that belongs to the phylum Sarcomastigophora and the subphyllum Mastigophora. Name this organism.

Trichomonas vaginalis

The above image is a protozoan that belongs to the phylum Sacomastigophora and the subphyllum Mastigophora. Name this organism:

Balantidium coli

The above image is a protozoan that belongs to the phylum Ciliophora. Name this organism:

Plasmodium vivax

The above image is a protozoan that belongs to the phylum Apicomplexa. This organism is in it's ring stage. Name this organism:

Plasmodium falciparum

The above image is a protozoan that belongs to the phylum Apicomplexa. Name this organism:

Saccharomyces cerevisiae

The above image is a non-motile fungi. This organism forms unicellular yest cells. Name this organism:

Penicillium notatum

The above image is a filamentous mold. Name this organism:

Aspergillus niger

The above image is a filamentous mold. Name this organism:

Ascaris lumbricoides

The above image is an egg of an organism that belongs to the phylum Nematoda. The organism grown from this egg is a roundworm. Identify what organism will hatch from this egg?

Ascaris lumbricoides

The above image is of an organism that belongs to the phylum Nematoda (rounds worms). Name this organism:

Enterobius vermicularis, pinworm

The above image is of eggs. The organism who these eggs belong to produce many thousands of eggs daily. The organism belongs to the phylum Nematoda (round worms). Name this organism:

Enterobius vermicularis, pinworm

The above image is of multiple organiams that belong to the phylum Nematoda (rounds worms). This organism is very small and reproduces very quickly. Name this organism:

Fasciola hepatica

The above image is of an organism that belongs the the phylum Platyhelminthes (flatworms), class trematodes (flukes). This organism is commonly called a liver fluke. Name this organism:

Schistosoma mansoni

The above image is of an organism that belongs to the phylum Platyhelminthes (flatworms), class trematodes (flukes). This organism's egg has a spike like projection on it. Name this organism:

Schistosoma mansoni

The above image is of an egg that belongs the an organism of the Platyhelminthe (flatworm) phylum, class Trematode (fluke). Name this organism:

Schistosoma japonicum

The above image is of an organism that belongs to the phylum Platyhelminthe (flatworm), class Trematode (fluke). This organism multiplies fastest of the two Schistosomes that were discussed in class. Name this organism:

Taenia solium

The above image is of an organism that belongs to the phylum Platyhelminthes (flatworms), class Cestodes (tapeworms). Name this organism:

Taenia solium

The above image is of an egg from an organism that belongs to the phylum Platyhelminthes (flatworms), class Cestodes (tapeworms). Notice the characteristic hooklets inside the egg. Name this organism:

Taenia solium

The above image is of an organism that belongs to the phylum Platyhelminthes (flatworms), class Cestodes (tapeworms). Notice the scolex and proglottids. Name this organism:

Serratia marcescens

The above organism grows red colonies when grown at the temp of 20-25 degrees. But when grown at 37 degress this organism grows milky white colonies. Name this organism:

Miccrococcus luteus

The above organism grows yellow colonies when grow on TSA regardless of the temperature it is grown at. Name this organism:

Microscope-Ocular Lenses

Top of microscope (binocular) magnifies 10x

Microscope- Objective Lenses

Scanning 4x X 10x= total 40 (red)
Low power 10x X 10x= total mag 100 (yellow)
High 40x X 10x= total mag 400 (blue)
Oil-Immersion 100x X 10x= total mag 1000 (white)

Microscope- nosepiece

allows 4 objective lenses to be turned into position and allows the microscope to be parfocal

Microscope- parfocal

ability to maintain focus when you change to a higher magnification

Microscope-arm

supports ocular and objective lenses

Microscope-base

supports entire microscope

Microscope-stage and mechanical stage control

holds slide and allows it to be moved over the surface of the stage

Microscope-condenser with condenser lens

aims light through the specimen

Microscope-substage adjustment knob

silver left side, this is for the condenser

Microscope-diaphram

regulates the amount of light passing through the condenser

Microscope-Coarse Adjustment Knob

allows for focusing

Microscope-Fine Adjustment Knob

allows for focusing- fine tunes focus

Microscope-Lamp Control Knob

round button at the bottom regulates the amount of light used

Microscope Inversion

put slide in but see it upside down, move slide left but it looks like it is moving right

Total Magnification

Ocular lens mag X objective lens mag

Resolution

ability to distinguish 2 separate points- makes it clear

Contrast

Difference in brightness/color- enable objects to be resolved more clearly ( this is why we stain to make contrast)

Diameter of Field

the distance across the entire visible field when looking through the ocular lenses. (1 round field)

Darkfield Microscope

Specimens appear bright against a dark background, no staining required-Live specimens may be observed

Phase Contrast Microscopes

special condenser lens, for improved visualization of internal details- no staining-Live specimen can be veiwed

Fluorescence Microscopes

Uses ultraviolet light source and fluorescent stains (called Fluorochromes)

Flurochromes

Some fluorochromes naturally attach to specific microorganisms (like mycobacterium tuberculosis)

Immuneofluorescence (Fluorescent antibody staining)

involves flourochromes that are chemically attached to antibodies known to bind specific microorganisms

Electron Microscopes

Uses Electron beams instead of a light source, specimen are placed in a vacuum inside the microscope so only dead specimens can be observed
Types: Transmission and Scanning

Transmission Electron Microscopes

Electrons pass through section of specimen(heavy metals are sometimes added to stain) usually black and white looks like a cross section- internal detail are clearly visible-since electron microscope only dead specimens

Scanning Electron Microscopes

Beam of electrons sweep over the surface of the specimen. Give 3D views of specimen's surface.since electron microscope only dead specimens

Scanning/tunneling; Atomic Force

Use tiny probes which interact the atoms on the specimen's surface. Information from the probes is computer-processed, producing a 3D image of specimen's surface- Live specimen's may be viewed- resolution so high that DNA or antibodies may be viewed.

Calculating diameter of field

millimeter in field from ruler-
1mm=1000um
diameter is in ums

diameter ratio equation

LPD = SPM
SPD LPM

5 ways to help with resolution

1. use shortwave length light source
2. diaphragm open
3. condenser up as high as poss.
4. stain to help with contrast
5. use immersion oil-keeps light from scattering

Standard smear

loopful of water on slide, spread in organism, let air dry then heat fix

2 reason to heat fix slides

1. Kills organisms
2. make them adhere to the slide

2 type of staining

Acidic-stains background
Basic-stains bacteria(opposite attract bacteria is acidic)

Acidic Stains

Nigrosine-black
India Ink-blue
Eosin-Red
These all stain backgrounds

Basic Stains

Crystal Violet- purple
Methylene Blue
Safranin, Carbol Fushsin-red
Malachite green
These stain the bacteria

Simple staining

Use 1 basic stain (colored portion is positively charges) to color cells. Allows for visualization of the cells size, shape, arrangement, and number.
standard smear, heat fix, stain Methylene Blue-1 minute, rinse
Bacillus Megaterium organism

Negative Staining

Use 1 acidic stain (colored portion is negatively charged) to color the background around cells. Allows visualization of size, shape, arrangement and number.
Drop of Nigrosine at end of slide
put Bacillus Magaterium in drop
spread with new slide
do NOT he

Capsule stain (nic)

Negative stain smear with
India Ink and Bacillus Megaterium then air dry and heat fix flood with crystal violet (basic stain) stand 1 min., rinse view

Differential Staining

Uses 2 diff. colored basic stains. Allows for discrimination of diff. types of cells
Gram Stain and Acid Fast Stain

What is Gram Staining

Steps to Gram Staining (cges)

Mixed standard smear of
E. Coli and Staphylococcus epidermidis, air dry heat fix
Crystal Violet (primary basic stain) 20 sec., rinse
Mordant (helps cell keep stain) Gram's iodine, 1 min.
95% ethanol decolorizer 10 sec. (makes gram- loss color) then rinse

What is acid fast staining

Steps to Acid Fast staining (hcab)

Standard smear of Staphylococcus Epidermidis and Mycobacterium Smegmatic, air dry and heat fix
On hot plate place slide and paper towel over weigh boat and heat while staining with CARBOLFUCHSIN (4-5 min., then cool and rinse
decolorize with ACID-ALCOHOL

Only 2 bacteria in the world that are red and acid fast in acid fast stain

Mycobacterium and Nocardia

Endospore Stain

Heat fixed bacterial smear of BACILLUS MEGATERIUM
stain with MILACHITE GREEN on hot plate for 3-5 min to force stain into the endospores, cool and rinse
Counterstain with SAFRANIN for 30 sec.

Bacteria used in all stain except acid-fast and gram staining

Bacillus Magaterium

Bacteria used in acid-fast and gram staining

Gram- E. coli- red (gram-) and Staphlyococcus Epidermitis- purple (gram+)
Acid-fast- Mycobaterium- red (acid-fast) and Staphyloccocus Epidermitis- blue (not acid-fast)

List 3 factors with influence clinical specimen quality

Proper collection, labeling, and Transport

Potential problem is specimen is not handled carefully

Pathogen may die
Overgrowth of normal flora may inhibit growth of pathogen
Presence of contaminants may interfere with identification of the pathogen

Proper Specimen collection for blood, throat swab and sputum.

blood-sterile equipment, degerm skin
throat-swab inflammed area back of throat
Sputum- deep within airway, no saliva

Proper specimen collection for wound, fecal and Gonococcal Specimens

Wound- needle aspirates from deep in the wound no surface swabs
Fecal- processed immediately to avoid temp and ph change
Gonococcal- Neisseria gonorrhoeae in very sensitive to cold NEVER REFRIDGERATE and must be incubated in a carbon dioxide-enriched envi

2 types of Antimicrobial agents and description
and other Antimicrobial agents

Broad Spectrum-work against variety or organisms
Narrow Spectrum- Wrok against certain kinds or organisms- ex penicillin (against gram +) organism
Others (antiviral, antifungal, antiprotozoan, and antihelminthic medications)

antibiotic that inhibit cell wall synthesis

Penicillin

antibiotic that inhibits protein synthesis

Tetracyclines

antibiotic that disrupts cell membrane function

Anti-fungal Drugs (zoles)

antibiotic that inhibit Nucleic Acid Synthesis

Cipro

antibiotic that interfere with enzyme function

sulfa drugs

What is the Kirby-Bauer Test for Antimicrobial Sensitivity

Uses Mueller Hinton agar, which is 4 mm thick, covered with E. coli or Staphylococcus aureaus with disks of antibiotics placed on plate and incubated for 16-18 hr.
zone of inhibition are measured in mm

What is Pseudomonas aeruginosa

Used in the demonstration plate of kirby-bauer sensitivity test
It's resistant to many antibiotics and found in the ICU, trauma, and burns-gives greenish pigment, gram-rod, resistant to everything but Cipro

Kirby-bauer sensitivity results for E. col

sensitive to all antibiotics except Penicillin (which is a narrow spectrum antibiotic)

Kirby-bauer sensitivity results for Staphylococcus Aureaus

Sensitive to everything but Nalidixic acid (NA-30)

Steps for Gram + unknowns testing

Streak Mannitol-salt agar (pink) plate to test for Salt-tolerance
Streak and stab blood agar plate to test for hemolysis

Gram + mannitol plate growth tells

if growth it is -salt tolerant (staph)
in no growth it is -not salt tolerant (Strep)

Gram + mannitol plate color

yellow-means that it ferments Mannitol and is Staphylococcus aureaus
Pink- means does not ferment mannitol
Staphylococcus epidermidis

Gram + blood agar plate beta, alpha and gamma hemolytic

Beta- white, agar dissapears-staph A
Gamma-no change in agar- Staph E
Beta- White, agar disappears- Strep Pyogenes
Alpha- Greenish Growth- Strep Pneumonia

What is a Catalase test?

put hydrogen peroxide on slide, put microb in it
bubbles- catalase Pos- Staph
no bubble- catalase neg- Strep

Coagulase

The coagulase test is used to differentiate Staphylococcus aureus from other staphs. The test uses rabbit plasma that has been inoculated with a staphylococcal colony. The tube is then incubated at 37 degrees Celsius for 1� hours. If negative then continu

What kind of test is a quick strep test

Antigen Detection
dissolve strep pyogenes in solution (specimen) and it moves up the test strip
Sample area has anti-Strep P antigens with red beads attached to them (from rabbits) they move up the dipstick and get trapped in the test area if strep is pre

Nucleic Acid testing?

Pathogens form patient specimens are identified using DNA or RNA probes. If DNA it is heated to separate the strands from each other. sequence for specific diseases are compared and if a sequence matches it has a fluorescent stain marking it for that dise

Staphyloslide agglutination test

uses antibodies to identify Staphylococcus A (which is normal flora on the skin but can cause infections in people)
Staph A have proteins on their surface that bind to human IgG antibodies
3 circles-
1 circle unknown- 1 drop of test latex(IGG antibodies)

gram- Bacilli unknown media used

Lactose broth- red test tube
Citrate agar- green test tube

Gram - Bacilli unknown result from Lactose broth (red)

stays red- doesn't ferment lactose
Serratia marcescens, Proteus vulgaris
turns yellow-ferments lactose
klebsiella pneumoniae, E. coli

Gram- Bacilli unknown result from Citrate agar

stays green-citrate negative
Proteus vulgaris, E.coli
turns blue-citrate positive
Klebsiella pneumoniae, serratia marcescens

What does citrate agar tell you

If the organism can use citrate as a carbon source, it turns blue if it is +

what are the 3 types of hemolysis and what do they look like

Beta-clear zone around colonies (destroys RBC)
Alpha-partial destruction, greenish/grey color around colony
Gamma-no destruction of RBC

what test tell you Staph A

MSA-growth, MSA-yellow, blood agar- beta
Staphyloslide aggulation test- clumps
Coagulase +

Enterotube II

multitest, used to distinguish gram - bacteria from one another

Standard Plate Count - Uses for enumerating (counting) the number of bacteria in a sample

1. Determines the Safety of a Food Product 2. The effectiveness of an antibiotic or disinfectant 3. The course of an infection 4. Deciding whether a beach should be closed due to fecal contamination

Standard Plate Count

Determine the number of bacteria in milk, various foods and water

First Step in Standard plate count

Diluting a sample to countable numbers and using a formula to calculate the number of bacteria in the original sample

Serial dilution

1g og soil in sterile water until getting between 30 - 300 colony forming units (CFU) to grow on a plate

CFU Formula

# of CFU (30 - 300) X the dilution factor (reciprocal of the dilution) = #CFU/g of soil.

Final answer from a CFU equation is

1. only an estimate 2. Only anerobic or facultative bacteria will grow under these conditions 3. Any anerobes in the sample will not be counted 4. Bacteria that needs special factors will not grow on general purpose media and will not be counted 5. Severa

Blanks

Sterile tubes or bottles containing measured amounts of sterile water

30 - 300 CFU - Number of CFU/Amount Plated X Dilution = CFU/g or ml

250 CFU - 250/0.1 X 10-5

Managing Microbial Growth

...

Bacteriostatic

Substances or conditions that inhibit growth of bacteria without killing them

Bactericidal

Substances or conditions that kill bacteria

Fungicide

lethal to fungi

Virucide

will kill or inactivate viruses

Sporicidal agent

will kill spores

Germicide

has activity against a wide range of microorganisms

Sterilization

destruction of all life forms. There is no life present in it or on it

Disinfection

destruction of vegetative pathogens but still contain living microbes

Binary Fission

Asexual reproductive method of most bacterial cells

Two Daughter Cells - Each with a complete set of chromosomes

During binary fission the cell replicates it's chromosome (DNA) and forms a septum the divides the cell into two of these___

Generation time

The amount of time it takes for a cell to replicate itself. Bacteria replication extremely variable eg. Escherichia coli divides every 20 minutes after 36 hours would cover the earth. Mycobacterium tuberculosis divides every 15 hours

Bacterial Growth Curve

the typical pattern followed by bacteria introduced into a new environment

Four phases of the Bacterial Growth Phase

1. Lag Phase 2. Logarithmic or Exponential phase 3. Stationary Phase 4. Decline or Death Phase

1. Lag Phase

Bacterial Growth Phase - little or no replication

2. Logarithmic or Exponential phase (log for short)

Bacterial Growth Phase - When the bacteria have adapted to the new environment and are metabolically active, reproducing exponentially

3. Stationary Phase

Bacterial Growth Phase - Due to the depletion of nutrient and the accumulation of waste products the death rate equals the rate of cell replication

4. Decline or Death Phase

Bacterial Growth Phase - As a nutrient becomes less available and the accumulation of waste products becomes more toxic the death rate exceeds the rate of depletion

When are the bacteria more easily inhibited or killed?

When they are in the logarithmic growth phase. Somewhat counterintuitive but many anti-microbial act by disrupting metabolism which is maximal during the log phase.

Bacterial Cells are harder to destroy in the stationary phase. Why?

Tend to have a lower metabolic rate, thicker cell walls and well developed capsules or endospores

Osmotic Pressure

...

Osmosis

movement of water across a semipermeable membrane

Osmotic Pressure

the force with which water moves across a membrane due to a concentration gradient

Hypertonic

if the surrounding environment contains high solute concentration relative to the inside of the cell

Plasmolysis

The loss of water from the cell will cause the cytoplasm to shrink away from the cell. The loss of water will inhibit growth of cells because so many metabolic reactions require water

Halophiles

Salt loving bacteria. When they can continue to grow in high salt concentrations - salting meats and fish and canning certain foods in a brine is an effective means of preserving

Hypotonic

if the surrounding environment has a low solute concentration and there will be a net movement of water into the cell.

Isotonic

if the solute concentration is the same inside and outside the cell

Gradient plates

a double-layered agar plate with a concentration gradient in the upper layer of agar

PH

...

Hydrogen Ion Concentration or PH

microorganisms have a specific range of this____which will influence it's growth

PH Optimum

PH at which an organism will grow best

PH Minimum and Maximum

PH above or below which will prevent their growth

Neutrophiles

neutral PH at which most bacteria will grow optimally

Alkalinophiles

inhabit alkaline soils and water and can continue to grow at a PH of 11.5 and above

Temperature

Enzyme activity and microbial growth is dependent on this

Temperature Range

different bacterial species have different optimal max and min _________ 20 to 30 degrees Celsius

Psychrophiles or Psychrophillic

Bacteria that can grow within a temperature range 0-20o C (cold loving)

Mesophiles

Bacteria that can grow within a temperature range 20o - 45o C (middle loving) Normal human body temperature

Thermophile or thermophilic

Bacteria that can grow within a temperature range 40o - 70o C (heat loving)

Hyperthermophiles or extreme thermophiles

80o C or higher and can grow in hot springs and geysers

Moist Heat Methods

most common and economic method of controlling microbial growth

Dry heat and moist heat

two different types of heat used to control microbial growth

Dry Heat method

kill microorganisms by denaturing proteins and oxidation (burning) of vital structural and metabolic chemicals

Direct Flaming

When the lip of the test tube or the loop of flamed this s what method of sterilization?

Drying Ovens

Circulation of hit air 171o C for 1 hour or 160o C for 2 hours to sterilize glassware and metal instruments, oils and powders

Incineration

(burning to ashes) used on a larger scale to dispose of contaminated materials in health care facilities and the stop the spread of infectious disease of infected cattle and poultry

Moist Heat Methods

kill microbes primarily by denaturing proteins and also damaging cell membranes and nucleic acids.

Moist Heat is more penetrating that dry heat

Can be effective at a lower temperature

Boiling water (100o C 15 to 30 min)

kills most vegetative cells but does not kill endospores. Does not result in sterilization but in disinfection for general purposes

Tyndallization (intermittent or Fractional Sterilization) Used to sterilize materials using 100o C steam for 30 mins but requires heating the material to be sterilized on three successive days and incubation of the material at 37o C between heat treatment

...

Autoclaving

uses steam heat under pressure and is one of the most reliable means of sterilization (15 PSI at 121o C for 30 mins)

Pasteruization

used in processing milk products - fruit juices - beer - wine. Does not result in sterilization but kills vegetative pathogens and some microorganisms involved in food spoilage

Heat Treatment principle

increasing temperature means decrease in time or lower the temperature means increasing the time for the same degree of disinfection

Based on the Heat Treatment Principle three types of pasteurization

low temperature (LTH) - 63o C for 30 min High Temperature short time (HTST) aka. Flash method - 72o C for 15 sec Ultra High Temperature (UHT) - raises the temperature from 74o C to 140o C rapidly and in less than 5 seconds lowers the temp back to 74o C

Ultraviolet Light

Electromagnetic radiation of several wavelengths is used to control microbial growth

Ionizing radiation

X-rays and gamma rays�are short in wave length - high in energy and very penetrating. Have sufficient energy to knock electrons free of atoms particularly water molecules forming free radicals

Ultraviolet Light

Electromagnetic radiation of several wavelengths is used to control microbial growth

Ionizing radiation

X-rays and gamma rays�are short in wave length - high in energy and very penetrating. Have sufficient energy to knock electrons free of atoms particularly water molecules forming free radicals. Directly impacts DNA causing lethal damage. Used to sterilize

Free Radicals

formed by ionizing radiation can cause lethal damage to cells

Gamma Radiation

used to sterilize various foods

Non-ionizing radiation

UV light is of longer wavelength (approx 10 to 400nm)

Wavelengths below 200nm

Absorbed by the air and are ineffective in controlling microbial growth except in a vacuum

UV light in wavelengths of 260 to 265 nm

corresponds to the wavelength that is absorbed optimally by DNA

Thymine dimmers

Disrupt both DNA replication and transcription and induced by UV light

UV light can be used for

1. Reduce the number of microbes on surfaces 2. Reduce the concentration of airborne microbes in operating rooms - isolation wards - areas of food preparation - sewage treatment plants

Dark Repair

Enzyme that repairs damage done to DNA from exposure to UV light - an endonuclease that can function without visible light and is able to remove pyrimidine dimmers

Light Repair or photorepair

a pyrimidine dimerase called photoreactivating enzyme which is activated by visible light in the blue and green spectrum (450 - 540nm)

gram stain reagents

crystal violet, iodine, acetone-alcohol, safranin

gram stain steps

primary stain, mordant, decolorizer, counter-stain

P-purple

gram positive

Red, hot, bad

gram NEGATIVE

a mordant is used for

adherence or enhancement of the primary stain

a primary stain is used to

color all cells the same

decolorizer is used to

remove stain from the negative cells, crystal violet particles are entrapped in the peptidoglycan of positive PURPLE cells

counterstain is used to

stain the gram negative RED cells

agar

is an extract from red marine algae used in culture media as a solidifying agent

agar liquifies at

100c

agar solidifies at

40c

transient microbes

are temporary guests that come and go

normal microbes

are permanent residents

lyme disease is caused by...

Borrelia burgdorferi

ocular lens

eyepieces of the microscope-remagnifies the image formed by the objective lens

body tube

the ocular lenses are attached to...transmits the image from the objectivelens to the ocular lens

arm

the main backbone of the scope, the best place to carry the microscope

objective lenses

primary lenses that manify the specimen

mechanical stage

holds the microscope slide in place

condenser

focuses light through the specimen

diaphragm

directly beneath the condenser, controls the amount of light entering the condenser

illuminator

the light source of the microscope, located in the base of the microscope

coarse focusing knob

large focusing wheel on the side

fine focusing knob

smaller focusing wheel on the side

base

foundation of the microscope

dental caries is caused by....

Streptoccus mutans.....strep-strip of gram positive PURPLE cocci

normal flora found in the nose, skin or places of high osmotic pressure...yellow clusters

Staphylococcus aureus

causative agent of Rocky Mountain Spotted Fever

Rickettsia rickettsii, gram negative rods or cocci that are obligate intracellular parasites

resolution

the microscopes ability to distinguish between two points at the particular resolution or higher

peritricous

multiple flagella all around the organisms body

atrichous

no flagella

amphitricous

flagella at the polar ends of an organism

lophotrichous

bundled tail or group of flagella at a polar end

cocci

round

bacillus

rod shaped

spiral

corkscrew`

cluster

group of cocci

chain

strip of cocci

Incubation temperature of cultures

35c

why 35c to incubate cultures

close to body temperature

biobags, sharps containers

disposal of hazardous waste

total magnification

ocular lens multiplied by the objective lens

field of vision

the area seen through a microscope

resolving power

the microscopes resolution is the ability of lenses to reveal fine detail or two points distinctly seperated

numerical aperture

the resolution is a function of the wavelength of light used and a characteristic of the lens system

refractive index

the amount light bends

focal point

as light rays pass through a lens they are bent to converge at one point

sperical aberration

fuzziness due to mutiple focal points

monochromatic light

use of one lightsource with one wavelength

parfocal

object is in focus in with one lens

complex media

media for which the exact chemical composition varies batch to batch

chemically defined media

a medium whose exact composition is known

nutrient broth

liquid complex media

nutrient agar

solid complex media

inoculated

introduction of microbes to media

turbid

cloudy

colony

population of cells that arises from a single bacterial cell

colony forming unit

unit of measure for microbes, a population of cells that arises from a single bacterial cell

flocculation

clumps of microbial cells

membrane

pellicle

sediment

settlement at the bottom of the liquid media

contaiminants

unwanted microbes in the media

aseptic technique

used in microbiology to exclude contaiminants

sterilized

free of all life

structural stains

used to identify and study the structure of bacteria

endospores

resting bodies

virulence

disease causing ability

Lowest Magnification

Used when first looking @ the slide. Either 4x or 10x

Spirochetes

Axial filaments; Cause lyme disease and syphillis

resolution

Microscopes ability to distinguish between 2 points greater than or equal to 0.2um

Total Magnification

multiply the magnification of the onjective lense usually 10x

Flagella Stain

spiral with polar flagella

Capule stain

Pink, looks splotchy

Gram Neg

Red

Gram Pos

Purple

purple

gram + (color)

pink

gram - (color)

crystal violet, gram's iodine, acetone alcohol, safranin

Gram stain colors

red

Acid Fast +

blue

Acid Fast -

Carbofuchsin, heat, acid alcohol, methylene blue

List Acid fast colors

red with green center

endospores present

red

no endospores present (color)

malachite green, heat, safranin

List Endospore stain colors

Endospores

_______-resistant to heat and chemicals, made of proteins

Capsules

structure that increases virulence, less vulnerable to phagocytosis

halo

capsule +

no halo

capsule -

gram + cocci

Mannitol Salt Agar gram??

Gram - rods

MacConkey- gram??

inhibited by salt

Mannitol salt- poor growth/ no growth

not inhibited by salt

Mannitol salt- good growth

mannitol fermentation produce acid

Mannitol salt- yellow

did not ferment mannitol

Mannitol salt- red

inhibited by crystal violet/ blue

MacConkey- poor growth/ no growth

not inhibited by crystal violet/ blue

MacConkey- good growth

produce acid from lactose fermentation

MacConkey- green or red

did not ferment lactose

MacConkey- colorless

Blood Agar- beta hemolysis

Complete clearing, organism is able to hemolyze RBCs

Blood Agar- alpha hemolysis

partial clearing (greening)

Blood Agar- Y hemolysis

organism does not hemolyze RBCs

catalase

detoxifies hydrogen peroxide

starch hydrolysis

bacteria's ability to hydrolyze starch with amylase

amylase

Hydrolytic enzyme uses water to breakdown starch

Gelatin hydrolysis

secretion of gelatinase by microbes to hydrolyze gelatin

secreted by microbes to hydrolyze gelatin

Gelatinase

Lipase test

Identify bacteria capable of producing the exoenzyme lipase

Clearing blue halo

Lipase +

No Clearing blue halo

Lipase -

Decarboxylase

enzyme that removes the decarboxyl group of an amino acid to yield an amine and CO2

Decarboxylase +

decarboxylase- pH becomes alkaline (purple)

Fermentation/Decarboxylase -

decarboxylase- yellow acidic

no decarboxylation

decarboxylase- no color change

gram - rod shaped

Coliform gram??

Coliform Test

ferment lactose with the production of acid & gas when incubated at 35-37C

Fermentation with acid and gas

Coliform test- yellow/ bubbles

Fermentation with acid and no gas

Coliform test- yellow/ no bubbles

No fermentation

Coliform test- red/ no bubbles

Degradation of peptone, alkaline end product

Coliform test- pink/ no bubbles

Eosin Methylene Blue Plate

isolate colonies, cause lactose fermentation bacteria to produce a purple color, black center, or metallic sheen.

Gram +

EMB- growth inhibited by EMB (poor growth/no growth)

Gram -

EMB- growth not inhibited by EMB (good growth)

Possible Coliform

EMB- pink and mucoid- organism ferments lactose with little acid production

Probable Coliform

EMB- Dark purple with or without green metallic sheen. Organism ferments lactose and or sucrose with much acid production

Non Coliform

EMB- growth is colorless- organism does not ferment lactose or sucrose

Phenol red turns yellow if acid is present

Carbohydrate Metabolism color?

Yellow with bubbles

Carbohydrate Metabolism- fermentation/ acid / gas production

Yellow no bubbles

Carbohydrate Metabolism- fermentation/ acid/ no gas production

red no bubbles

Carbohydrate Metabolism- no fermentation, no acid, no gas production (color)

pink no bubbles

Carbohydrate Metabolism- degradation of peptone, alkaline end product

Citrate Test

ability of an organism to use citrate as its sole carbon source (instead of utilizing glucose)

Citrate utilized

Citrate test- Blue, small growth

Citrate utilized

Citrate test- no color change, growth

Citrate not utilized

Citrate test- no color change, no growth

MR Test

Test indicates an organism's ability to produce large amounts of ACID from glucose

VP test

Test used to detect the presence of acetoin

MR

Methyl Red

VP

Potassium hydroxide and a-naphthol

mixed acid fermentation

MR Test- Red

no mixed acid fermentation

MR Test- no color change (stays yellow)

Acetoin

VP Test- Red

no Acetoin

VP Test- no color change

SIM Test

Test the production of sulfide reduction, indole, and motility

Black

Sulfer reduced to sulfide which reacts with Fe+ and becomes ____

reddish, trypthophan is broken down into indole and pyruvate

Indole is detected by Fe+ (color)

Kovac's reagent

use of ______ to breakdown tryptophan into indole and pyruvate

70s ribosomes

Tetracycline targets _____

Phenol Red

Water Coliform Test- dye?

Phenol Red

Carbohydrate Metabolism- dye?

Lugol's Iodine

Starch hydrolysis- dye?

Kovac's Reagent

SIM test- dye?

Hydrogen Peroxide

Catalase Test- dye?

Bromthymol blue

Citrate Test- dye?

Eosin Methylene Blue

EMB test- dye?

Resident Flora/ Normal Flora

Colonize on body. Symbiotic relationship.

Commensalism

One benefits, other indifferent.

Mutualism

Both benefit. A lot of this occurs in the stomach and intestines.

Transient Flora

May be on any body for short period of time. They don't colonize on body.

Opportunistic Pathogens

Doesn't cause disease in healthy individuals, under certain conditions can cause disease. Exs: yeast infection - Candida albicans.

Pathogens

Disease causing.

Microbial Antagonism

Typically normal flora inhabit and colonize the body and prevent other pathogens from colonizing on the body. Microbes competing against one another for space and nutrients.

Chemically Defined Medium

ExaExact chemical component is known.

Complex Media

Exact chemical component is not known, varies slightly from batch to batch - usually some type of extract used. Exs: Nutrient broth or agar, TSA - tryptic soy agar, meat extracts, peptone - partially digested proteins.

What is Agar?

Extracts from marine algae. Main function is a solidifying agent. Most microbes can't degrade it so it remains solid.

What are some characteristics of Agar?

Agar doesn't have any nutrients. Agar is only used as a solidifying agent. There is no nutritional value. Solid at 40�C and liquid at 100�C

What are the building blocks of life?

Carbon, Nitrogen, Protein

Autoclaving

Steam sterilization under pressure.

Colony

All bacteria are genetically identical.

Soaps and Detergents

Function as surfactants. Decreases surface tension of water. Emulsifies fats and oily film on skin. Either kills bacteria or more importantly removes them from skin.

Surfactants

Wetting agents decrease surface tension.

Biocide

High concentration gradient. Targets membrane and cytoplasm.

Bacteriostatic

Low concentration gradient. Targets fatty acid synthesis.

How do soaps target fatty acid synthesis?

Binds to ENR (ethyl-acyl carrier protein reductose enzyme). Then binds to NAD (nicotramole adenne dinucleotide). This new structure can't participate in fatty acid synthesis. Fatty acid needed for cells to grow.

Diagram of what is happening when fatty acid synthesis is disabled

Tricloson ----binds to--- ENR---which binds to---NAD. Now the entire structure of Tricloson-ENR-NAD can't participate in fatty acid synthesis.

Different types of resistance for soaps and detergents.

Mutations in gene for ENR-> ENR won't bind to tricloson.
Overexpressed gene for ENR -> more ENR some still function.
Certain bacteria pumps out tricloson (Psuedomonas aeniginosa).
Alternative gene for ENR that doesn't bind to tricloson (Bacillus).

Hand Sanitizers

Alcohols (ethanol-drinking, isopropanol-rubbing). Effective against bacteria, fungi, and viruses. Denature proteins and disrupt cytoplasmic membranes.

What is optimal % of ethanol?

62-70% of ethanol.

Why isn't 100% ethanol optimal?

Because the sanitizer would dry up completely before you are able to kill the bacteria completely. Also, water helps facilitate it faster.

What is most predominant type of bacteria in the mouth?

Streptococcus.

What are two examples of streptococcus bacteria?

S. mutans and S. sanguinis

What do streptococcus produce?

Sticky polysacchrides from sucrose.

How does streptococcus hydrolyze sucrose?

nsucrose=glucose(little)n + nfructose
Glucose + energy (polymerization via dextransucrose). Dextran fructose fermented to produce lactic acid.

Dextran

Sticky carbohydrate chains that surround bacteria. Enables bacteria to adhere to the surface of the mouth (teeth).

Plaque

Masses of bacteria, dextran, debris adhering to teeth (acts as net that traps sugars and food particles).

Lactic acid

Erodes tooth enamel -> caries.

Where do streptococci grow?

Exposed dentin and tooth pulp.

Sugarless Candies

Usually contain sugar alcohols (sorbitol, mannitol). Can't be converted into dextran (no glucose product). Can still be fermented.

Snyder Agar

Contains dextrose (glucose), Bromcresol green (pH indicator).

Bromcresol Green

pH indicator. Neutral = green Acidic = yellow Basic = blue. Other ingredients are yeast extract, NaCl, etc.

Fructose

Fructose supplies energy and by-product is lactic acid.

What microscope do we use in Micro Lab?

Compound Brightfield Microscope

Objective Lens

Lens closest to object (4, 10, 40, 100x)

Ocular Lens

Closest to eye (10x)

How do you figure out total magnification?

objmag x ocu.mag

Brightfield

Light passes through speciment. Specimen appears "shadowy" through a bright background.

Resolution

Clarity of an image -> the smaller the better.

Limit of Resolution (D)

Measurement of how far 2 points must be before the microscope can view them as separate. The smaller D is the better.

Equation for Limit of Resolution

D=?/NA condenser + NA objective lens

?

Wavelength of light used. Shorter wavelength more ideal.

NA

numerical aperture. Lens ability to "capture" light. Lager NA more ideal.

Refractive Index

Speed of light in substance. Air ~1, Oil ~1.5, Glass ~1.5. Oil matches refractive index of glass so light doesn't "bend". Glass to air means light will "bend" and get lost.

How do images appear in a microscope?

It is an upside down mirror image!

Pure Culture

Single kind of microbe. Required to study growth, pathogenicity, metabolism, antibiotic susceptibility.

What are the 3 current methods for isolation?

Streak plate, spread plate, pour plate.

Streak Plate

Stream mixed sample over solid medium in a petri plate. Theoretically bacteria fall off the loop over different areas on the agar and they form a colony.

Spread Plate

Sample diluted, spread over solid medium surface use spreading rod.

Pour Plate

Diluted sample mixed with agar, poured into empty petri dishes. Colonies form in and on plate.

CFU

Colony Forming Units

CFU Equation

# of Colonies/Amount plated X Dilution

Aseptic Technique

Used to exclude contaminants.

Sterilized Culture Media

Autoclave

What are the 3 different ways to culture?

Broth, Slant, Agar Deeps

Broth

Easy to transport, large population of bacteria. Fast media easily accessible gets nutrients fast but stop growing after 48 hrs.

Slant

Easier to store and transport than plates. Small accessible surface, nutrients seep up, maintains cultures for long periods of time.

Agar Deeps

Used to grow bacteria that may require Oxygen, also used to determine motility. Bacteria will swim away from point of inoculation. They are a semi-solid.

What can we learn from looking at bacteria?

Morphology/shape, Size, Arrangement

Why do we use stains and dyes?

Bacteria appears transparent so we use stains and dyes to increase the contrast.

Simple Stains

Only one reagent is used.

Direct Stains

Stains the bacteria directly.

Negative Stains

Stains the background, the bacteria is unstained.

What are the 3 kinds of stains?

Simple stains, direct stains, negative stains.

How does staining work?

The stains are charged colored ions (chomophores) in solution?

Chomophores

Charged colored ions.

Cationic

#NAME?

Basic Stain

(+) charge on the ion (cationic). Ex: Methylene Blue

Anionic

#NAME?

Acidic Stain

(-) Charge on the ion (anionic). Ex: India Ink.

What is the bacterial cell wall charged?

Slightly negative.

Basic stains...

Stain the bacteria directly.

Acidic stains...

Stain around the bacteria.

Smear

Thin film of bacteria on a slide.

Fixing

Making the bacteria adhere to the slide (coagulating proteins). Also kills the bacteria.

Heat Fix

Pass smear through Bunsen Burner.

Chemically Fix

Cover in 95% Methanol.

Negative Stain Procedure

The culture is mixed with a stain then smeared across the slide (feathering). No heat fixing is used. This is helpful because the cells are not distorted and more accurately show bacteria morphology and size.

What techniques do we use to observe live organisms?

Wet Mount and Hanging Drop.

Wet Mount

A drop of culture, then a glass coverslip placed over it.

What are advantages to a wet mount?

Fast, Cheap, Disposable.

What are disadvantages to a wet mount?

Dries fast, so can't be observed long.

Hanging Drop

Special slide with depression used, culture drop on inverted coverslip, then placed on depressed slide with petroleum jelly seal.

What are advantages to a hanging drop?

Doesn't dry out as fast. You can observe longer.

What are disadvantages to a hanging drop?

Special slides used, labor intensive, and expensive.

Motility

Self-directed movement of an organism via flagella, cilia, or psuedopod.

Flagella

Very long hair-like.

Cilia

Very short hair-like.

Psuedopod

Foot like.

Chemotaxis

Organism can "sense" chemicals and move towards it or away from it.

Why are chemotaxis beneficial to bacteria?

Help for pathogenicity since it can evade the hosts defense and spread throughout the body.

Brownian Motion

A vibrations motion caused by water molecules colliding with the microorganism.

Differential Stains

Involve more than one stain. Allows us to differentiate between various bacteria or bring out specific structures.

Gram Stain

Useful for identifying and classifying bacteria. Based on bacteria cell wall properties. Allows us to differentiate between Gram (+) and Gram (-) bacteria.

Bacterial Cell Wall

Composed of peptidoglycan.

Peptidoglycan

Consists of repeating disaccharides linked with peptides.

Two types of disaccharides

N-acetylglutamine (NAG) and N-acetylmutamic acid (NAM)

Gram (+) Bacteria

Several layers of peptidoglycan. Very thick and rigid. Contains teichoic acid.

Gram (-) Bacteria

Thin layer of peptidoglycan. Contains outer membrane (lipo-polysaccharides, lipoproteins, phospholipids) with porin.

Porin

Channels allow for certain molecules to enter.

What are the steps in a Gram Stain?

Prepare smear. Let air dry completely. Add crystal violet for 1 min then rinse. Add iodine for 1 min then rinse. Add 95% EtOH for 5-15 seconds then rinse. Add safranin for 1 min then rinse. Blot dry and view.

What is the function of crystal violet?

Primary Stain.

What color is Gram (+) when crystal violet is added?

Purple

What color is Gram (-) when crystal violet is added?

Purple

What is the function of Iodine?

Mordant (binding agent).

What color is Gram (+) when iodine is added?

Purple

What color is Gram (-) when iodine is added?

Purple

What is the function of 95% Ethoyl alcohol?

Decolorizing agent.

What color is Gram (+) when EtOH is added?

Purple

What color is Gram (-) when EtOH is added?

Clear/Colorless

What is the function of Safranin?

Counterstain or Secondary Stain

What color is Gram (+) when Safranin is added?

Purple

What color is Gram (-) when Safranin is added?

Red/Pink

What color is Gram (+) bacteria?

Purple!!!

What color is Gram (-) bacteria?

Red/Pink

What are some reasons for inaccurate results?

Destained too long, destained too short, cells too old.

Gram Variable

If you are not able to tell Gram rxn.

Clinical application of Gram Staining?

Gram rxn tells us about bacteria so we know which drug to prescribe.

Penicillin

Interferes with linking peptide cross bridges. Weakens cell wall and ultimately causes cell lysis. Has a much better effect on Gram (+) bacteria.

Acid Fast Bacteria

Retain the primary stain even when treated with acid alcohol. Cell walls contain mycolic acid. Includes Mycobacterium and Nocardia species which include human pathogens as M. tuberculosis and M. Leprae.

Mycolic Acid

Waxy lipid, makes cell permeable to most stains.

How does acid fast staining work?

Bacteria are first stained with Carbolfuchsin. Acid alcohol is then added. Bacteria that are not acid fast are quickly destained. Since the carbolfuchsin has phenol in it and phenol is more soluble in fat than acid alcohol. The mycolic acid will retain th

Carbolfuchsin

Red dye containing phenol.

Non acid-fast bacteria are stained

blue

Acid-fast bacteria are stained

red

Is mycolic acid impermeable to methylene blue?

Yes

Vegetative Bodies

Bacteria that are actively metablizing.

What bacteria produce endospores?

Clostridium and Bacillus species. C. tetani, C. botulism, B. anthracis, C. difficile

Endospores

Thick outer layer of keratin. Resistant to heat, drying, detergents, harsh environment, UV, etc. Not actively metabolizing. Smaller and more compact than vegetative bodies.

How do endospores form?

The vegetative body compacts DNA and forms endospores. The vegetative body disintegrates. Endospores are formed via sporogenesis.

Why do endospores form?

This occurs in harsh environments as a survival mechanism. If nutrients decrease and there is a chance the vegetative body might die then endospores are formed.

When do endospores turn back into vegetative bodies?

When environment is more appealing the endospores germinate and are back to being vegetative bodies.

Are endospores a reproductive mechanism?

No, it is a survival mechanism. 1 veg. body = 1 endospore.

How do we do a smear for endospores and vegetative bodies?

A standard heat-fix smear is prepared. Melachite green added and gently heated over flame. Since endospores have thick later of keratin, heating is required for stain penetration. The Malachite Green was away, then counterstain Safranin is added.

What does Safranin stain?

Stains vegetative cell bodies not the endospore.

Capsules

Gelatinous coat surrounding certain bacteria (glycocalyx). Composed of uncharged polysaccharides. Size is influenced by media its cultured in. Increases bacteria virulence.

How do capsules increase bacterial virulence?

Helps evade phagocytosis, WBC can't hold onto a slippery capsule. Helps bacteria attach to cause infection. Functions as an osmotic barrier, prevents dehydration.

How do we do a stain to see capsules?

Prepare a standard negative stain. This will stain the background, neither cell nor capsule will be stained. Add a basic stain (crystal violet) over the prep. This will stain only the cell body. Since capsule has no charge the basic dye will not stain it.

Eukaryotes

Contain a nucleus.

Heterotrophic

Cannot produce it's own Carbon source, must obtain from environment.

Saprophytic

Obtain nutrients from dead matter.

Yeast & Molds

Eukaryotes, heterotrophic, some are saprophytic, some are pathogenic. Cell walls composed of chitin. Prefers a low pH and a high concentration of sugar. Special media used to culture.

What is the special media used to culture yeast and molds?

Sabouraud Agar

Sabouraud Agar

Contains peptone, glucose with a high low pH to inhibit bacterial growth.

Yeast

Unicellular. Round or ovoid. Asexual reproduction/budding. Can be sexual as well as asexual.

Dimorphic

Yeast & Mold. Can switch due to environmental cue. Ex: E. albicans, Ustilago mayous

Molds

Multicellular, filamentous hyphae, asexual or sexual reproduction via spores.

How is fungi categorized and identified?

Structually vs. metabolically like bacteria.

Hyphae

Classified by function.

Vegetative Hyphae

Grows laterally or underneath surface, absorbs nutrients.

Reproductive Hyphae

Grows vertically, gives rise to spores. Can be septated or non-septated.

Septated

Hyphae divided into compartments,

Several hyphae

Mycelium

What are the 2 types of asexual spore formation?

Sporangiospores and Conidiospores

Sporangiospores

Spores formed in a sac (sporangium)

Conidiospores

Free spores not enclosed by sac.

What are 3 different types of media?

Selective media, enrichment media, and differential media.

Selective Media

Contains chemicals that prevent the growth of unwanted bacteria without inhibiting the growth of the desired bacteria. EX: Mannitol Salt Agar (MSA) and eosin Methylene Blue (EMB)

Enrichment Media

Usually liquid media contains chemicals that enhance the growth of the desired bacteria. Other bacteria will grow but increases the growth of desired bacteria.

Differential Media

Contains various nutrients that allow us to distinguish one bacteria from another based on how they metabolize, or change media with a waste product.

Mannitol Salt Agar (MSA)

NaCl - 7.5%. Media is selective for salt tolerant bacteria. Mannitol -1% differentiating properties. Differentiates between Mannitol fermenters and non-mannitol fermenters. Phenol Red - 0.025% pH indicator

Phenol Red

0.025% pH indicator.

Pink/Red on Phenol Red

Neutral

Yellow on Phenol Red

Acidic

Shocking Pink on Phenol Red

Basic

What color will something be if it is a mannitol fermenter?

Yellow

What makes MSA differential?

Mannitol - 1%

What is the MSA differential between?

Mannitol fermenters and non-mannitol fermenters.

What makes the MSA selective?

NaCl - 7.5%

What is the MSA selective for?

Salt tolerant bacteria.

Eosin Methylene Blue (EMB)

Lactose: 0.5% sugar, makes it differential. Eosin & Methylene Blue: dyes that make media selective. Gram (+) can't grow: selective for Gram (-) bacteria. Differential for lactose fermenters (purple, green metallic sheen) and non-lactose fermenters (pink/t

What makes EMB selective?

Eosin and Methylene Blue dyes.

What is EMB selective for?

Gram (-) bacteria.

What makes EMB differential?

Lactose - 0.5% sugar

What is EMB differential for?

Lactose fermenters and non-lactose fermenters.

What color are lactose fermenters on EMB?

Purple, green metallic sheen.

What color are non-lactose fermenters on EMB?

Pink, translucent.

MacConkey Agar

Selective and differential.

What makes MacConkey Agar selective?

Bile salts.

What is MacConkey Agar selective for?

Gram (-) bacteria.

What makes MacConkey Agar differential?

Lactose

What is MacConkey Agar selective for?

Lactose fermenters and non-lactose fermenters.

What do lactose fermenters on MacConkey Agar look like?

Red, opaque colonies.

What do non-lactose fermenters on MacConkey Agar look like?

Translucent

Phenylethyl Alcohol Agar (PEA)

Phenylethyl alcohol inhibits DNA synthesis in Gram (-) bacteria.

What makes PEA selective?

Phenylethyl alcohol.

What is PEA selective for?

Gram (+) bacteria.

Blood Agar

Defibrinated sheep's blood. NaCl- 0.5% minimize spontaneous hemolysis.

What are the different types of hemolytic reactions?

Alpha, Beta, Gamma

Alpha Hemolysis

Green and cloudy around colony. Partial destruction to water from bacteria. A=Alright

Beta Hemolysis

Complete hemolysis, clear zone around colony. B=Bad

Gamma Hemolysis

No hemolysis. G=Good

Catabolism

Chemical rxns that release energy from decomposition of organic molecules.

What are 3 types of carbohydrates?

Monosaccharides, oligosaccharides, and polysaccharides,

Monosaccharides

Simple sugars.

Oligosaccharides

2-20 monosaccharides (disaccharides are most common).

Polysaccharides

8 or more monosaccharides.

Oxidative Catabolism

Requires the presence of oxygen. End products are carbon dioxide and water.

Fermentative Catabolism

Doesn't require oxygen but can occur in its presence. End products are usually organic acids, some bacteria can produce gas.

Oxidative Fermentative Test (OF)

Used to determine if an organism is oxidative or fermentative for a carb.

What is used in the OF Test?

Semi-solid agar deep with high concentration of carbs and low concentration of peptone. 2 tubes used one is open to air and the other air is kept out of.

What is the pH indicator for the OF Test?

Phenol Red

How do you read results for the OF Test?

In order to be considered oxidized one tube must be yellow on top and red on the bottom while the other tube must be all red. Yellow indicates acid production.

In the OF test what does it mean if both tubes turn yellow?

Glucose was fermented.

In order for both tubes to be fermented what must happen?

Both tubes must turn yellow.

What is medium used for Sugar Fermentation Test?

Pr-glucose PR- lactose or PR-sucrose

How do you read results for SF test?

If yellow is seen in Durham tube than sugar is fermented. If gas bubble is seen then gas is produced.

What happens if sugar is fermented in SF test?

Turns yellow

What happens if gas is produced in SF test?

Bubble in Durham tube.

Electron Acceptors

Molecules that combine with e- liberated during metabolic processes.

Reduction

Gain of e-

Fermentative

Organic e- acceptor

Respiration

Inorganic electron acceptors. Ex: Oxygen is final electrons acceptor in aerobic respiration.

Cytochromes C

Carry e- to oxygen. There are 4 different classes of cytochromes (one is cytochrome C) The oxidase test, tests for the presence of the enzyme e oxidase.

Catalase

An enzyme produced by most aerobic bacteria which breaks down Hydrogen Peroxide into Water and Oxygen.

What does the Catalase test test for?

The presence of coenzyme catalase.

Enzymes

Proteins that catalyze biochemical rxns.

Endoenzymes

Function inside the cell.

Exoenzymes

Released from cell to catalyze rxns outside the cell.

Hydrolytic Enzymes

Exoenzymes break down substrates with addition of water. EX: Amylase - hydrolyzes starch -> smaller carbs.

What is the exoenzyme being tested for in the Casein Hydrolysis Test?

Caseinase

What is media for Casein Hydrolysis Test?

Skim milk agar.

What is theory behind Casein Hydrolysis Test?

It is protein catabolism. Bacteria that produce caseinase will hydrolyze the caseinase around the bacteria colony or streak resulting in a clear zone.

What is the exoenzyme being tested for in the Starch Hydrolysis Test?

Amylase.

What is media in Starch Hydrolysis Test?

Starch Agar Plate (NA supp. with starch).

What is theory behind Starch Hydrolysis Test?

It is carb catabolism. Bacteria that produce amylase will hydrolyze. The starch surrounding colony or streaky. Iodine (a starch binding agent) is added to detect presence of absence of starch around bacteria streak.

What is the exoenzyme being tested in the Gelatin Hydrolysis Test?

Gelatinase.

What is the media used in the Gelatin Hydrolysis Test?

Nutrient gelatin deep (NG).

What is the theory behind the Gelatin Hydrolysis Test?

It is protein catabolism. Gelatin is solid at RT. Bacteria that hydrolyze gelatin via gelatinase will break peptide bonds converting media from solid into liquid.

What 4 tests make up the IMViC?

I-Indole, M-Methyl Red, V- Voges-Proskauer, C-Citrate.

What does the Indole Test test for?

Production of tryptophanase.

What does the Methyl Red test for?

Acid production from glucose

What does the Voges-Proskauer test for?

Production of acetoin from glucose.

What does the Citrate test for?

Na citrate as only carbon source.

What is the purpose of Indole Production Test?

Detect bacteria that have tryptophanase.

What is the media used in the Indole Production test?

Tryptone broth.

What is theory behind Indole Test?

Some bacteria produce tryptophan so an enzyme that can hydrolyze tryptophan into indole, pyruvic acid, and ammonia.

How do we test for indole production?

Adding Kovacs reagent to detect indole production.

Energy & Gram Neg.

What do we need to determine in microbes by doing metabolic activity assays

Phenol red

What is the indicator in both the phenol red broth test and the urease test

pH

What does the indicator phenol red indicate in the phenol red broth test

Gas

What is the Durham tube used to collect during the phenol red broth test

Acid fermentation

What process are we testing for in the phenol red broth test

Yellow

What color(s) is a positive phenol red broth test (acidic)

Bright pink

What color is a negative phenol red broth test (basic)

Orange

What color(s) is a negative phenol red broth test (neutral)

Enterobacteria

What medical application (microbe) is the phenol red broth, phenylalanine deaminase, gelatin hydrolysis, and SIM tests used to detect that is gram negative (most common)

Hydrogen peroxide

What do you mix bacteria with for a catalase assay

Enzyme catalase

What are we testing for by doing the catalase assay

Bubbles

What is a positive result of the catalase assay

No bubbles

What is a negative result of the catalase test

Staph

What common microb will it indicate if you get a positive catalase assay

Strep

What common microb will it indicate if you get a negative catalase assay

Ferric chloride

What is the reagent used in the Phenylalanine Deaminease Test

Green

What color(s) does ferric chloride turn in the Phenylalanine Deaminease Test

Amino acids

We are looking for ________________ in Phenylalanine Deaminease test and the SIM test to see if there is energy

Protein

Amino acids are the same thing as

Phenylalanine Deaminease Test