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699 terms

Final Set - Lab Final

STUDY
PLAY
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 streptococci are susceptible to bacitracin.
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 heat fix, just air dry because you are not adding a stain that has to be rinsed.
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
Safranin stain (counter basic stain) 20 sec.
gram - end up red (E.coli)
gram + end up purple (Staphylococcus Epidermidis)
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 3-5 sec.
Counter stain with METHYLENE BLUE 30 sec.
End up with Red-(acid-fast)mycobacterium and Blue Staph
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 environment
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 1½ hours. If negative then continue incubation up to 18 hours.
If positive (i.e., the suspect colony is S. aureus), the serum will coagulate,[2] resulting in a clot (sometimes the clot is so pronounced that the liquid will completely solidify).
If negative, the plasma remains liquid.
Coagulase pos- means Staph A
Coagulase neg- means other staph
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 present because the antigens bind to the antibodies for strep lining up the beads. The control antibodies further up attach to the red beads making a line.
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 disease
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)
2 circle staph.epi- neg control- 1 drop control latex
3 circle staph A- pos control-1 drop of test latex (IGG)
should clump in 3, not in 2
will only clump in 1 if it is staph A
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. Several bacterial cells are aggregated (clumped together) and will only form one CFU
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 treatments
...
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 heat labile (destroyed by heat) products such as plastic syringes and surgical gloves
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=λ/NA condenser + NA objective lens
λ
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
+ charge on the ion
Basic Stain
(+) charge on the ion (cationic). Ex: Methylene Blue
Anionic
- Charge on the ion.
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 the stain even after the acid alcohol rinses. Methylene Blue is used as a counter-stain.
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. Capsule will appear as a "halo" around the stained bacterium against a dark background.
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/translucent).
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
Yellow is the indication that ____________ test is negative
Negative
The Phenylalanine Deaminease Test medical application most commonly detect Enterobacteria which is gram
Iodine
What is the reagent in the starch hydrolysis test
H2O
The starch hydrolysis test is to see if there is an addition of _______ to breakdown startch so it can be used as energy
Glowing
A positive starch hydrolysis test shows and clear zone surrounding the growth and is
Dark brown
A negative starch hydrolysis test is the color(s) of
E. coli
What comon gram negative bacteria does the starch hydrolysis test for
Nitrogen
The urease test is to determine the waste bacteria of __________ that we excrete in urine
Ammonia
We are testing for what is a byproduct of urease in the urease test
Red or pink
A positive result in the urease test would be the color(s)
Basic
Is ammonia (a byproduct of urease) basic or acidic
No color
A negative result color(s) of the urease test, SIM:indole & SIM: Hydrogen sulfide production test is
Proteus
The medical application that the urease test is for is usually for urinary tract pathogens from the gram negative genus
Gelatin
The gelatin hydrolysis assay is used to detect for the breakdown of
Gelatinase
The only way the breakdown of gelatin happens in the gelatin hydrolysis assay is if it contains the enzyme
Liquid
The results of a positive gelatin hydrolysis test is
Solid
The results of a negative gelatin hydrolysis test is
Kovac's
What is the reagent used in the SIM: indole production
Tryptophan
What does Kovac's reagent break down in the SIM iodole test
Pink
A positive SIM:indole test wouls be the color(s)
Sulfer
The SIM: Hydrogen sulfide production test is the breakdown of amino acids using
Black
A positive SIM: Hydrogen sulfide production test is the color(s)
Metal ions
What turns black in the positive result of the SIM: Hydrogen sulfide production test
Cloudy
A positive SIM: Motility test would be
Growth
A negative SIM: Motility test would only have _______ at the stab line
Carbohydrates
Acid fermentation is the same as the ability to ferment ____________ in the phenol red broth test
6.8 - 7.4
Neutral pH
< 6.8
Acidic pH
> 7.4
Alkaline pH
color change & gas formed
What are the signs of fermentation
Yes
For fermentation, can you have acid without gas
No
For fermentation, can you have gas without acid
Water and gas
The two things the catalase test converts hydrogen peroxide into is
O2
What is being released in the catalase test that forms bubbles
Polysaccharide
What type of molecule is starch
Bacillus cereus
What comon gram positive bacteria does the starch hydrolysis test for
Ammonia & CO2
The molecule urea can be metabolized by c ertain bacteria into two things
Urease
Ammonia & CO2 contain the enzyme ______
For energy
Why would a bacterium want to hydrolyze gelatin.
Candy
What product do we usally find the enzyme gelatinase in
Deamination
The ability of a bacteria to remove the amine group from the amino acid phenylalanine is called