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?
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?
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?
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?
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?
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?
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?
This is a differential stain used to detect the presence and location of spores in bacterial cells. What type of stain is this?
The above test is used to determine what?
What type of agar is used in this test?
Would this be considered a positive or negative starch hydrolysis test?
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?
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?
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?
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?
The above image shows what test?
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?
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?
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?
The above test is used to differentiate organisms on their ability to hydrolyze urea using the enzyme urease. What is this test called?
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?
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?
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?
Does the above MSA plate select for gram-positive of gram-negative organisms?
What organism does the above medium favor the growth of?
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?
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?
The above organism is growing on blood agar, what type of hemolysis is seen?
The above organism is growing on blood agar, what type of hemolysis is seen?
The above organism is growing on blood agar, what type of hemolysis is seen?
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.
The above bacitracin susceptibility test is run on what medium?
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.
The above plate is yellow before inoculation. What type of agar is this?
What is the indicator in MacConkey agar?
MacConkey agar inhibits the growth of gram-negative or gram-positive organisms?
MacConkey agar is used to isolate and differentiate members of the Enterobacteriaceae based on the ability to _______________________ ____________________ (turns the medium pink).
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?
EMB selects for gram-negative of gram-positive organisms?
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:
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?
The above organism is a protozoan that belongs to the phylum Sarcomastigophora and the subphyllum Sarcodina. Name this organism
The above image is a protozoan that belongs to the phylum Sarcomastigophora and the subphyllum Mastigophora. Name this organism.
The above image is a protozoan that belongs to the phylum Sacomastigophora and the subphyllum Mastigophora. Name this organism:
The above image is a protozoan that belongs to the phylum Ciliophora. Name this organism:
The above image is a protozoan that belongs to the phylum Apicomplexa. This organism is in it's ring stage. Name this organism:
The above image is a protozoan that belongs to the phylum Apicomplexa. Name this organism:
The above image is a non-motile fungi. This organism forms unicellular yest cells. Name this organism:
The above image is a filamentous mold. Name this organism:
The above image is a filamentous mold. Name this organism:
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?
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:
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:
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:
The above image is of an egg that belongs the an organism of the Platyhelminthe (flatworm) phylum, class Trematode (fluke). Name this organism:
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:
The above image is of an organism that belongs to the phylum Platyhelminthes (flatworms), class Cestodes (tapeworms). Name this organism:
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:
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:
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:
The above organism grows yellow colonies when grow on TSA regardless of the temperature it is grown at. Name this organism:
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)
allows 4 objective lenses to be turned into position and allows the microscope to be parfocal
ability to maintain focus when you change to a higher magnification
supports ocular and objective lenses
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
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
put slide in but see it upside down, move slide left but it looks like it is moving right
Ocular lens mag X objective lens mag
ability to distinguish 2 separate points- makes it clear
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)
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
Uses ultraviolet light source and fluorescent stains (called Fluorochromes)
Some fluorochromes naturally attach to specific microorganisms (like mycobacterium tuberculosis)
involves flourochromes that are chemically attached to antibodies known to bind specific microorganisms
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
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)
Nigrosine-black India Ink-blue Eosin-Red These all stain backgrounds
Crystal Violet- purple Methylene Blue Safranin, Carbol Fushsin-red Malachite green These stain the bacteria
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
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
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
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
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
antibiotic that inhibits protein synthesis
antibiotic that disrupts cell membrane function
Anti-fungal Drugs (zoles)
antibiotic that inhibit Nucleic Acid Synthesis
antibiotic that interfere with enzyme function
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
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, 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)
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
1g og soil in sterile water until getting between 30 - 300 colony forming units (CFU) to grow on a plate
# 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
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
Substances or conditions that inhibit growth of bacteria without killing them
Substances or conditions that kill bacteria
lethal to fungi
will kill or inactivate viruses
will kill spores
has activity against a wide range of microorganisms
destruction of all life forms. There is no life present in it or on it
destruction of vegetative pathogens but still contain living microbes
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___
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
movement of water across a semipermeable membrane
the force with which water moves across a membrane due to a concentration gradient
if the surrounding environment contains high solute concentration relative to the inside of the cell
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
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
if the surrounding environment has a low solute concentration and there will be a net movement of water into the cell.
if the solute concentration is the same inside and outside the cell
a double-layered agar plate with a concentration gradient in the upper layer of agar
Hydrogen Ion Concentration or PH
microorganisms have a specific range of this____which will influence it's growth
PH at which an organism will grow best
PH Minimum and Maximum
PH above or below which will prevent their growth
neutral PH at which most bacteria will grow optimally
inhabit alkaline soils and water and can continue to grow at a PH of 11.5 and above
Enzyme activity and microbial growth is dependent on this
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)
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
When the lip of the test tube or the loop of flamed this s what method of sterilization?
Circulation of hit air 171o C for 1 hour or 160o C for 2 hours to sterilize glassware and metal instruments, oils and powders
(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
uses steam heat under pressure and is one of the most reliable means of sterilization (15 PSI at 121o C for 30 mins)
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
Electromagnetic radiation of several wavelengths is used to control microbial growth
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
Electromagnetic radiation of several wavelengths is used to control microbial growth
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
formed by ionizing radiation can cause lethal damage to cells
used to sterilize various foods
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
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
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)
_______-resistant to heat and chemicals, made of proteins
structure that increases virulence, less vulnerable to phagocytosis
gram + cocci
Mannitol Salt Agar gram??
Gram - rods
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
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
detoxifies hydrogen peroxide
bacteria's ability to hydrolyze starch with amylase
Hydrolytic enzyme uses water to breakdown starch
secretion of gelatinase by microbes to hydrolyze gelatin
secreted by microbes to hydrolyze gelatin
Identify bacteria capable of producing the exoenzyme lipase
Clearing blue halo
No Clearing blue halo
enzyme that removes the decarboxyl group of an amino acid to yield an amine and CO2
decarboxylase- pH becomes alkaline (purple)
decarboxylase- yellow acidic
decarboxylase- no color change
gram - rod shaped
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
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.
EMB- growth inhibited by EMB (poor growth/no growth)
EMB- growth not inhibited by EMB (good growth)
EMB- pink and mucoid- organism ferments lactose with little acid production
EMB- Dark purple with or without green metallic sheen. Organism ferments lactose and or sucrose with much acid production
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
ability of an organism to use citrate as its sole carbon source (instead of utilizing glucose)
Citrate test- Blue, small growth
Citrate test- no color change, growth
Citrate not utilized
Citrate test- no color change, no growth
Test indicates an organism's ability to produce large amounts of ACID from glucose
Test used to detect the presence of acetoin
Potassium hydroxide and a-naphthol
mixed acid fermentation
MR Test- Red
no mixed acid fermentation
MR Test- no color change (stays yellow)
VP Test- Red
VP Test- no color change
Test the production of sulfide reduction, indole, and motility
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)
use of ______ to breakdown tryptophan into indole and pyruvate
Tetracycline targets _____
Water Coliform Test- dye?
Carbohydrate Metabolism- dye?
Starch hydrolysis- dye?
SIM test- dye?
Catalase Test- dye?
Citrate Test- dye?
Eosin Methylene Blue
EMB test- dye?
Resident Flora/ Normal Flora
Colonize on body. Symbiotic relationship.
One benefits, other indifferent.
Both benefit. A lot of this occurs in the stomach and intestines.
May be on any body for short period of time. They don't colonize on body.
Doesn't cause disease in healthy individuals, under certain conditions can cause disease. Exs: yeast infection - Candida albicans.
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.
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
Steam sterilization under pressure.
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.
Wetting agents decrease surface tension.
High concentration gradient. Targets membrane and cytoplasm.
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).
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?
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.
Sticky carbohydrate chains that surround bacteria. Enables bacteria to adhere to the surface of the mouth (teeth).
Masses of bacteria, dextran, debris adhering to teeth (acts as net that traps sugars and food particles).
Erodes tooth enamel -> caries.
Where do streptococci grow?
Exposed dentin and tooth pulp.
Usually contain sugar alcohols (sorbitol, mannitol). Can't be converted into dextran (no glucose product). Can still be fermented.
Contains dextrose (glucose), Bromcresol green (pH indicator).
pH indicator. Neutral = green Acidic = yellow Basic = blue. Other ingredients are yeast extract, NaCl, etc.
Fructose supplies energy and by-product is lactic acid.
What microscope do we use in Micro Lab?
Compound Brightfield Microscope
Lens closest to object (4, 10, 40, 100x)
Closest to eye (10x)
How do you figure out total magnification?
objmag x ocu.mag
Light passes through speciment. Specimen appears "shadowy" through a bright background.
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
Wavelength of light used. Shorter wavelength more ideal.
numerical aperture. Lens ability to "capture" light. Lager NA more ideal.
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!
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.
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.
Sample diluted, spread over solid medium surface use spreading rod.
Diluted sample mixed with agar, poured into empty petri dishes. Colonies form in and on plate.
Colony Forming Units
# of Colonies/Amount plated X Dilution
Used to exclude contaminants.
Sterilized Culture Media
What are the 3 different ways to culture?
Broth, Slant, Agar Deeps
Easy to transport, large population of bacteria. Fast media easily accessible gets nutrients fast but stop growing after 48 hrs.
Easier to store and transport than plates. Small accessible surface, nutrients seep up, maintains cultures for long periods of time.
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.
Only one reagent is used.
Stains the bacteria directly.
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?
Charged colored ions.
+ charge on the ion
(+) charge on the ion (cationic). Ex: Methylene Blue
- Charge on the ion.
(-) Charge on the ion (anionic). Ex: India Ink.
What is the bacterial cell wall charged?
Stain the bacteria directly.
Stain around the bacteria.
Thin film of bacteria on a slide.
Making the bacteria adhere to the slide (coagulating proteins). Also kills the bacteria.
Pass smear through Bunsen Burner.
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.
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.
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.
Self-directed movement of an organism via flagella, cilia, or psuedopod.
Very long hair-like.
Very short hair-like.
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.
A vibrations motion caused by water molecules colliding with the microorganism.
Involve more than one stain. Allows us to differentiate between various bacteria or bring out specific structures.
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.
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.
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?
What color is Gram (+) when crystal violet is added?
What color is Gram (-) when crystal violet is added?
What is the function of Iodine?
Mordant (binding agent).
What color is Gram (+) when iodine is added?
What color is Gram (-) when iodine is added?
What is the function of 95% Ethoyl alcohol?
What color is Gram (+) when EtOH is added?
What color is Gram (-) when EtOH is added?
What is the function of Safranin?
Counterstain or Secondary Stain
What color is Gram (+) when Safranin is added?
What color is Gram (-) when Safranin is added?
What color is Gram (+) bacteria?
What color is Gram (-) bacteria?
What are some reasons for inaccurate results?
Destained too long, destained too short, cells too old.
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.
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.
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.
Red dye containing phenol.
Non acid-fast bacteria are stained
Acid-fast bacteria are stained
Is mycolic acid impermeable to methylene blue?
Bacteria that are actively metablizing.
What bacteria produce endospores?
Clostridium and Bacillus species. C. tetani, C. botulism, B. anthracis, C. difficile
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.
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.
Contain a nucleus.
Cannot produce it's own Carbon source, must obtain from environment.
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?
Contains peptone, glucose with a high low pH to inhibit bacterial growth.
Unicellular. Round or ovoid. Asexual reproduction/budding. Can be sexual as well as asexual.
Yeast & Mold. Can switch due to environmental cue. Ex: E. albicans, Ustilago mayous
Multicellular, filamentous hyphae, asexual or sexual reproduction via spores.
How is fungi categorized and identified?
Structually vs. metabolically like bacteria.
Classified by function.
Grows laterally or underneath surface, absorbs nutrients.
Grows vertically, gives rise to spores. Can be septated or non-septated.
Hyphae divided into compartments,
What are the 2 types of asexual spore formation?
Sporangiospores and Conidiospores
Spores formed in a sac (sporangium)
Free spores not enclosed by sac.
What are 3 different types of media?
Selective media, enrichment media, and differential 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)
Usually liquid media contains chemicals that enhance the growth of the desired bacteria. Other bacteria will grow but increases the growth of desired bacteria.
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
0.025% pH indicator.
Pink/Red on Phenol Red
Yellow on Phenol Red
Shocking Pink on Phenol Red
What color will something be if it is a mannitol fermenter?
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?
Selective and differential.
What makes MacConkey Agar selective?
What is MacConkey Agar selective for?
Gram (-) bacteria.
What makes MacConkey Agar differential?
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?
Phenylethyl Alcohol Agar (PEA)
Phenylethyl alcohol inhibits DNA synthesis in Gram (-) bacteria.
What are the different types of hemolytic reactions?
Alpha, Beta, Gamma
Green and cloudy around colony. Partial destruction to water from bacteria. A=Alright
Complete hemolysis, clear zone around colony. B=Bad
No hemolysis. G=Good
Chemical rxns that release energy from decomposition of organic molecules.
What are 3 types of carbohydrates?
Monosaccharides, oligosaccharides, and polysaccharides,
2-20 monosaccharides (disaccharides are most common).
8 or more monosaccharides.
Requires the presence of oxygen. End products are carbon dioxide and water.
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?
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?
What happens if gas is produced in SF test?
Bubble in Durham tube.
Molecules that combine with e- liberated during metabolic processes.
Gain of e-
Organic e- acceptor
Inorganic electron acceptors. Ex: Oxygen is final electrons acceptor in aerobic respiration.
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.
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.
Proteins that catalyze biochemical rxns.
Function inside the cell.
Released from cell to catalyze rxns outside the cell.
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?
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?
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?
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.