Lab Practical Review Flashcards
Spore forming pathogens are most likely Gram + or Gram -? Rods or Cocci?
Gram + Rods
Gram Stain Steps
Drop of distilled water on slide first
Than mix in bacteria
Allow to air dry
Heat fix over burner (3-4 passes over flame)
Once completely dry begin the staining portion
Crystal Violet - 30 seconds
RINSE
Gram’s Iodine (Mordant) - 30 seconds
RINSE
Decolorizing Alcohol - 10 drops/until its clear
RINSE
Safranin Counterstain - 30 seconds
RINSE
Use Bibulous paper to dry
- In the Gram stain, a bacterial smear is dried and then heat-fixed to cause it to adhere to the glass slide. It is then stained with crystal violet dye, the primary stain, which is rinsed off and replaced with Gram’s iodine. The iodine acts as a mordant - that is, it binds the dye to the cell. The smear is then decolorized with alcohol and counterstained with safranin. Gram (+) organisms take on the crystal violet stain which gets locked in by the Gram’s iodine. They are dark blue/purple in color and are dense. Gram (-) organisms with their fatty cell walls do not take on the first two steps of the Gram stain, but the decolorizing alcohol allows the counterstain to absorb. They are red and more translucent.*
What color does a Gram + pathogen stain in a gram stain?
- Blue.
- Gram (+) organisms take on the crystal violet stain which gets locked in by the Gram’s iodine.
- They are dark blue/purple in color and are dense.
What color does a Gram - pathogen stain a gram stain?
- Red.
- Gram (-) organisms with their fatty cell walls do not take on the first two steps of the Gram stain, but the decolorizing alcohol allows the counterstain to absorb.
- They are red and more translucent.
What does a Gram Stain tell us?
- Bacterial Shape & Arrangement
- If the bacteria is Gram (+) or Gram (-)
- Gram (+) cell walls contain peptidoglycan which is easy to stain and hard to decolorize. - Gram (-) cell walls are made up of phospholipids which are difficult to stain, but easy to decolorize
Do you heat fix a Gram Stain Slide?
YES
What does a Negative Stain tell us?
Morphology only. The shape & arrangement of the microbe
Do you heat fix a Negative Stain?
NO
What type of stain is this?
Gram Stain
What type of stain is this?
Negative Stain
What does an Endospore Stain tell us?
Determines whether the microbe is a spore forming bacteria or not
What is the most common spore forming bacteria?
Gram + Rods
Explain the Negative Stain steps
Procedure:
1. Flame and cool your loop and place a loopful of India ink in the center of a clean microscope slide
2. Flame and cool your loop and mix a small amount of bacteria into the stain
3. Mix and spread the bacteria out
4. Let AIR DRY - Do NOT heat fix as this will distort the shape
5. examine under oil immersion
Explain the Endospore Stain steps
- Add water to a beaker and bring to a boil
2 Prepare a smear on a clean slide and heat fix - Place your slide on the beaker. Place a piece of paper towel over smear - this will help prevent the dye from running off the slide
- Place drops of Malachite green dye on the paper towel and steam for 5 minutes. Continue to add stain to prevent the dye from drying on the slide
- Decolorize with water for about 30 seconds. The vegetative cells lose the dye, but the endospores retain the dye
- Counterstain with safranin for 30 seconds. Rinse with water. Blot dry with bibulous paper
- Observe under oil immersion. The endospores appear green and the vegetative cells appear pink
When a bacteria is grown on separate agar plates at different temperatures (degree celsius) what does that tell us?
This experiment shows us that different bacteria have different temperatures in which they prefer to grow.
What can we observe from these plates?
From left To right:
10 C
20 C
30 C
40 C
50 C
1. E. coli
2. Pseudomonas fluorescens
3. Bacillus stearothermophilus
Define: Obligate Aerobe
Grows only in the presence of oxygen
Define: Obligate Anaerobe
Will not grow in the presence of oxygen.
What type of stain & bacteria shape is seen?
Negative Stain - cocci
What type of stain & bacteria shape is seen?
Negative Stain - rods
Special Notes to consider to Improve Your Gram Stain?
- Gram (+) organisms can lose their ability to retain the crystal violet complex as they age. This can cause the smear to appear Gram variable - with both colors present. It is interesting to note that Gram (+) organisms can appear Gram (-), but Gram (-) organisms never appear Gram (+).
- Gram (-) cocci are really, really, really, really, really, really, really rare.
- The most important step to the Gram stain is the decolorizing alcohol. You can decolorize too much and cause a Gram (+) organism to appear Gram (-).
- Thick smears do not stain well. Spread your smears out and look towards the thinner edges.
What type of stain & bacteria shape is seen?
Gram Stain. Gram (+) rods
What type of stain & bacteria shape is seen?
Gram Stain. Gram (+) cocci
What type of stain & bacteria shape is seen?
Gram Stain. Gram (-) rods
DEFINE: Endospore
Highly resistant metabolically inactive cell types
DEFINE: Germination
Endospores reacting to favorable conditions and becoming metabolically active
DEFINE: Vegetative cell
Metabolically active form of bacteria
DEFINE: Capsule
Gelatinous protective coating formed by SOME bacteria -difficult to stain
DEFINE: Capsule
a protective gelatinous coating produced by some bacteria. Capsules are difficult to stain, and may not show up on a Gram stain
Explain the Capsule Stain steps
- Make a suspension of the organism in a drop of water on a clean slide
- Put a drop of India ink next to the drop
- Carefully lower a coverslip over the two drops so that they mix together. There should be a gradient in the concentration of the ink
- Examine the slide under oil immersion and find a field where you can see the cells surrounded by a halo in a black background (it looks like static on a television)
- Dispose of the slide in the sharps container
Endospore stain
- Prepare a smear on a clean slide and heat fix 2. Add water to a beaker and bring to a boil
- Place your slide on the beaker. Place a piece of paper towel over smear - this will help prevent the dye from running off the slide
- Place drops of Malachite green dye on the paper towel and steam for 5 minutes. Continue to add stain to prevent the dye from drying on the slide
- Decolorize with water for about 30 seconds. The vegetative cells lose the dye, but the endospores retain the dye
- Counterstain with safranin for 30 seconds. Rinse with water. Blot dry with bibulous paper
- Observe under oil immersion. The endospores appear green and the vegetative cells appear pink
- Record results
- Perform a Gram stain and compare to the endospore stain. Note that in a Gram stain, endospores do not stain and the cells appear to have holes in them
What type of stain & bacteria shape is seen?
Gram stain of a spore forming Gram (+) rod
What type of stain & bacteria shape is seen?
Gram stain of a spore forming Gram (+) rodSpore
What type of stain & bacteria shape is seen?
Endospore stain of Vegetative cells
E. coli dislikes what temps?
Cold & Hot temps
What is the point of Aseptic Technique?
The two goals of aseptic technique are to prevent contamination of your culture with organisms from the environment and to prevent the culture from contaminating you or others.
Streak Plate technique is also referred to as?
Streak to Isolate
Explain Streak Plate Steps
- Label the agar plate on the bottom with your initials and date.
- Divide the plate into three sections with a T as diagrammed.
- Flame and cool your loop
- Gently roll the culture between your hands to mix. Flame the mouth of the tube. Aseptically remove a loopful of the culture, flame the mouth of the tube, recap the tube and place back in the holder. You will only use the bacteria culture once!
- Holding your loop as you would a pencil, spread the bacteria on section 1 of the plate by streaking back and forth. The more streaks, the better chance of isolated colonies. As you work, partially cover the petri dish with the cover to minimize environmental contamination. Use a gliding motion and avoid gouging your agar.
- This is the important part! Do NOT get more bacteria! Flame and cool your loop!
- Streak section 2 by starting in section 1 and pull into section 2. Do this 3-4 times and spread into section 2.
- Flame and cool your loop!
- Streak section 3 by starting in section 2 and pull into section 3. Do this 3-4 times and spread into section 3.
- Flame and cool your loop.
- Incubate the plates upside down in the colored holder found at your table. Incubate at room temperature, 25°C.
The purpose of streak plates are?
- Streak to Isolate.
Purpose is to take a mixture of bacteria and dilute it enough so that each individual colony will contain only one type of bacteria.
Most samples taken in a vet clinic will be a mixture, so the streak plate is an essential skill.
This technique is used to obtain a pure culture.
What materials do you need for a steak plate?
- Trypticase soy agar plate (TSA Plate)
- Broth culture containing a mixture of two organisms
- Loop & buns burner
Define: Compound microscope
A microscope with more than one lens system
Define: Condenser
A structure located below the microscope stage that contains a lens for focusing light on the specimen as well as an iris diaphragm
Define: Immersion oil
Oil placed on a slide to minimize refraction of the light entering the lens
Define: Iris diaphragm
An adjustable opening that regulates the amount of light illuminating the specimen
Define: Magnification
The microscope’s ability to optically increase the specimen size
Define: Refraction
The bending of light as it passes from one medium to another
Define: Resolution
The smallest separation that two structural forms must have in order to be distinguished optically as separate
What magnification are most organisms viewed under the microscope?
most organisms require the use of the oil immersion lens (100X) + Oculars which are 10x so total of 1000x magnification
Why do we use immersion oil?
Immersion oil prevents light refraction and allows for better magnification and resolution.
E. coli oxygen preference is classified as?
Facultative Anaerobe
What does this test show us?
Tubes Labeled:
1. Micrococcus luteus
2. Clostridium sporogenes
3. E. Coli
This is an oxygen slant test showing us that only the obligate aerobe (M. luteus) and facultative anaerobe (E. coli) could grow on the slants because of the oxygen present in the room
What are the oxygen results?
Obligate Aerobe
What are the oxygen results?
Facultative Anaerobe
What are the oxygen results?
Aerotolerant Anaerobe
What are the oxygen results?
Obligate anaerobe
Osmotic Pressure Measures?
SALT CONCENTRATION
S. aureus osmotic pressure preference is?
likes salt and can grow even at 11% salt concentration.
E. coli osmotic pressure preference is?
does not tolerate high levels of salt and only grows at 2% salt concentration.
E. coli does not tolerate high levels of salt and only grows at 2% salt concentration.
S. aureus likes salt and can grow even at 11% salt concentration.
Liquid Media = Broth
Semi-solid Media = Agar
Plates (Petri dish)
Deep (test tube, agar solidified straight across)
Slant (test tube, agar solidified at an angle to create more surface for bacteria growth)
Media Type: Broth
Liquid Media
Media Type: Semi-solid Media
Agar -
- Plates (Petri dish)
- Deep (test tube, agar solidified straight across)
- Slant (test tube, agar solidified at an angle to create more surface for bacteria growth)
Name the media type
Semi-solid Media - Agar Plate
Name the media type
Semi-solid Media - Agar Deep Tube
Name the media type
Semi-solid Media - Agar Slant
Name the media type
Liquid Media = Broth
What are the 3 types of growth media and what is the purpose of different growth media types?
Complex, Selective and Differential Media
- Growth media can have different ingredients and purposes. Using certain media can inhibit the growth of some bacteria while promoting the growth of other bacteria. Added ingredients can help when identifying organisms or determining pathogenicity.
Media Type: Complex medium
Also termed undefined medium. A medium in which the exact amounts of chemical components and their composition are unknown. Tend to promote the growth of many types of bacteria
Media Type: Chemically defined media
A synthetic medium composed of inorganic salts and usually a carbon source such as glucose
Media Type: Differential medium
Medium permitting certain organisms to be distinguished from others by the appearance of their colonies. Useful in isolating and identifying bacteria
Media Type: Selective medium
Medium formulated to permit the growth of certain bacteria but not others. Useful in isolating certain bacteria
Staphylococcus epidermidis is what type of bacteria?
Gram (+) cocci
Enterobacter aerogenes is what type of bacteria?
Gram (-) rod
Pseudomonas aeruginosa is what type of bacteria?
Gram (-) rod
E. coli is what type of bacteria?
Gram (-) rod
TSA media is what type of media? & support what type of growth?
- complex medium
- supports general growth
- Gram positive and negative pathogens
Glucose salts media is what type of media? & support what type of growth?
- chemically defined medium
- Only organisms that can make all their cellular components from glucose and inorganic salts are able to grow on it
- Promotes growth of Gram (-) organisms
Eosin-methylene blue agar (EMB) agar is what type of media? & support what type of growth?
-selective medium Also differential because it contains the sugar lactose.
- Promotes the growth of Gram (-) enteric rods
- Organisms that can ferment lactose produce purple colonies.
- Those that cannot produce white or very light pink colonies
Phenyl-ethyl alcohol agar (PEA) media is what type of media? & support what type of growth?
- selective media
- Promotes the growth of Gram (+) organisms
Endo media is what type of media? & support what type of growth?
- selective & Differential with lactose
- Promotes Gram (-)
- lactose fermenters grow red colonies
- non-fermenters grow colorless colonies
What is significant about E. coli growth on Eosin-methylene blue agar (EMB) agar?
E. coli ( a lactose fermenter) produces dark purple colonies and a distinctive metallic green sheen
Mannitol salts media is what type of media? & support what type of growth?
- selective due to high salt concentration
- Promotes the growth of “halophilic” (salt loving) organisms
- Mannitol fermenters will change the color of the agar to yellow
MacConkey media is what type of media? & support what type of growth?
- selective
- promotes G(-)
- Differential with lactose
- lactose fermenters grow bright pink colonies
Blood agar media is what type of media? & support what type of growth?
- rich complex media that supports general growth.
- Helpful in determining pathogenicity.
- If bacteria can utilize red blood cells for energy, they are more pathogenic.
Beta hemolysis:
complete utilization of red blood cells for energy - clear zone around colony growth
Alpha hemolysis:
partial utilization of red blood cells for energy - greenish haze around colony growth
Gamma hemolysis:
no utilization of red blood cells for energy - no change to media
Alpha hemolysis
Beta hemolysis
Gamma hemolysis
Label the 4 quadrants
1 = alpha growth
2 = gamma growth
3 = beta growth
4 = alpha growth
Label the 4 quadrants
- The bacteria in quadrant number #2
was the only gram (+) bacteria of the
four, and clearly would not grow on the
MacConkey plate. - Bacteria #2 was S. epidermidis (+) cocci which doesn’t grow well in a MacConkey agar.
- The bacteria in quadrant #1 is clearly a “lactose fermenter” as indicated by its pinkish color.
What does this experiment tell us?
Mannitol fermenters will change the color of the agar to yellow
What does this experiment tell us?
In quadrants 3 & 4, the bacteria have
Grown a pinkish shade, indicating “lactose
fermenters”.
What does this experiment show on PEA?
Quadrant 2 is S. aureus, G(+) cocci.
Note that P. aeruginosa (3) was able to grow, but it is capable of using almost anything for its carbon source for E, so often times will grow when least expected.
(4) = Contaminant bacteria (probably a drip of the S. aureus)
The numbers are counter-clockwise
In this picture.
Explain what’s happening
The top is (1.) E. Coli.
which has that shimmery, metallic green,
indicative of a “coliform”
bacteria.
- S. aureus, G(+) cocci – does not grow
The bacteria in quadrants 3 & 4 are
a pinkish color, indicating “lactose
fermenters”
All three G(-) organisms grow well
Note #2 S. epidermidis (G(+) cocci that likes higher salt concentrations)
Does not like the high glucose content, so does not grow
What can we note about the growth in this TSA plate?
All four bacteria grow well on TSA
Serial Dilutions measure how?
Studies involving the analysis of materials such as food, water, or milk require quantitative evaluation (getting a bacterial count).
We will be using the direct plate count which requires serial dilution of the bacteria in order to get countable numbers. Because we cannot tell just by looking at a sample how much a sample should be diluted before we have countable numbers, we do several plates at once.
Creating serial dilutions and plate counts
Only count plates with how many CFU?
30-300 CFU
Serial dilution calculation is used to determine?
To determine the number of bacteria in 1ml of a solution
Ultraviolet (UV) light works how?
By killing microorganisms by acting on their DNA, causing mutations.
UV light does not penetrate surfaces and will not go through ordinary plastic or glass.
It is only useful for killing organisms on surfaces and in the air.
Explain a Ultraviolet (UV) light test?
Create bacterial lawns with a variety of bacteria (non-sporeforming and sporeforming)
Place the plates under the UV light. Place the Petri dish lid half on, half off the agar. The covered part will act as the control because UV radiation does not affect penetrate most plastics.
What test is being run& what is happening?
- UV light excerise
- In the bottom row, the B. cereus (a spore forming bacteria) was quite resistant to UV exposure until it was exposed for 15 minutes.
KIRBY-BAUER METHOD OF ANTIMICROBIAL SUSCEPTIBILITY Steps:
•Mueller-Hinton agar plate which– supports the growth of a variety of organisms
•Create a bacterial lawn
•Add infused paper discs to the lawn
•Incubate upside down
•Measure zones of inhibition
•Measure diameter of zone in mm
•Typically, the biggest zone “wins”
•Refer to published efficacy data with antibiotics
•Antibiotics, disinfectants, essential oils are used in this experiment
ZONES OF INHIBITION refer to which test?
KIRBY-BAUER METHOD OF ANTIMICROBIAL SUSCEPTIBILITY TEST
Catalase Test tell us what?
•Catalase: An enzyme found in most aerobic organisms that breaks down H2O2 to water and oxygen
•Bubble formation = Positive Catalase
•No bubble formation = Negative Catalase
the Oxidase Test tells us what?
•The oxidase test is used to identify bacteria that produce cytochrome c oxidase, an enzyme of the bacterial electron transport chain
•Immediate color change = Positive Oxidase
•No immediate color change = Negative Oxidase
Phenyl Fermentation Tubes Test tell us?
•Determines if a bacteria can ferment various simple sugars (lactose, glucose, sucrose, etc)
•Can provide a confirmation to MacConkey agar results for lactose fermentation
•Contains a Durham tube which is an inverted empty tube that captures any gas production – shows as a bubble
•Contains pH indicator: color change from alkaline pH (red) to acidic pH (yellow)
•Color change from red (uninoculated) to yellow indicates fermentation and acid production positive (+)
•Recorded as Acid/Gas: -/-; +/- (A/-); +/+ (A/G)
Simmons Citrate is used to?
•Used to determine if bacteria can utilize sodium citrate as a carbon source
•Contains pH indicator, if a bacteria can utilize sodium citrate, this will create an alkaline pH
•Color change from a green to brilliant blue = Positive
Urease test tell us?
• If the organism produces the enzyme urease, it can break the urea into ammonia and CO2.
• This raises the pH of the medium, turning it bright pink.
•Organisms are grown on agar containing urea and a pH indicator
Methyl Red test tell us?
• If the organism is capable of fermentation and the end products are acetic or lactic acids, the pH will lower to acidic range and the test will be positive.
•The methyl red reagent is designed to detect organisms that can overcome a phosphate buffer and can lower the pH of an environment, making it favorable for survival of that bacteria.
•MR-VP broth can be used for both MR and VP tests (must use separate tubes, one for each test)
Voges-Proskauer test is used for?
• for bacteria that use a different pathway for fermentation. The end products of these organisms are mostly alcohols.
• Also uses buffered peptone glucose broth (MR-VP broth).
•Voges Proskauer reagents are designed to detect bacterial fermentation that produces “acetoin.”
•Must let sit at least 20 minutes before calling the test negative
SIM media is what type of media? & tests for?
• is an agar deep that tests for H2S production, indole, and motility.
- A straight needle stab inoculation is used. SIM media is incubated at 25°C.
•H2S production results in black coloration of the media
•Indole: Some organisms have an enzyme that cleaves the amino acid tryptophan, producing indole. After incubation, Kovacs reagent is added. A red ring forms on top of the media if the organism is indole positive
•Motility: Cloudiness spreading from the point of inoculation indicates the organism in motile
Define: Catalase
An enzyme found in most aerobic organisms that breaks down H2O2 to water and oxygen
What test is being used?
KIRBY-BAUER METHOD OF ANTIMICROBIAL SUSCEPTIBILITY
Label the arrows
What test is being used? & what are the results?
Catalase test.
Bubble formation = Positive Catalase
No bubble formation = Negative Catalase
What test is being used? & what are the results?
Catalase test.
Bubble formation = Positive Catalase
No bubble formation = Negative Catalase
What test is being used? & what are the results?
oxidase test
Immediate color change = Positive Oxidase
No immediate color change = Negative Oxidase
What test is being used? & what are the results?
oxidase test
Immediate color change = Positive Oxidase
No immediate color change = Negative Oxidase
What test is being used? & what are the results?
Color change from red (uninoculated) to yellow indicates fermentation and acid production (+)
What test is being used? & what are the results?
Simmons Citrate.
Color change from a green to brilliant blue = Positive
What test is being used? & what are the results?
Urease tear.
If positive it bright pink.
What test is being used? & what are the results?
Methyl Red Test.
Positive for fermentation if the colors changes to Red.
What test is being used? & what are the results?
Methyl Red Test.
Positive for fermentation if the colors changes to Red.
What test is being used? & what are the results?
Voges-Proskauer test.
Must wait 20 mins for results
Red color on top shows positive for bacterial fermentation that produce Acetoin
What test is being used? & what are the results?
Voges-Proskauer test. Must wait 20 mins for results
Red color on top shows positive for bacterial fermentation that produce Acetoin
What test is being used? & what are the results?
SIM Media.
Indole positive = red ring forms on top of the media
Motility= Cloudiness spreading from the point of inoculation indicates the organism in motile
Sulfur production = black discoloration
What test is being used? & what are the results?
SIM Media.
Indole positive = red ring forms on top of the media
Motility= Cloudiness spreading from the point of inoculation indicates the organism in motile
Sulfur production = black discoloration
