Lab Final Exam Flashcards
Identify this part of the microscope and its function
Arm
Stability and Support
Identify the part of the microscope and its function
Base
Stability and Support
Identify the part of the microscope and its function
Coarse Adjustment Knob
For Scanning power focus of the specimen
Identify the part of the microscope and its function
Condenser
Focuses light onto the specimen
Identify the part of the microscope and its function
Diaphragm
Channels light through the aperture
Identify the part of the microscope and its function
Fine adjustment knob
High power focusing of the specimen
Identify the part of the microscope and its function
Beams light through the diaphragm
Identify the part of the microscope and its function
Mechanical Stage
Supports the specimen being viewed
Identify the part of the microscope and its function
Objective Lenses
Collects light from the sample for delivery to the ocular lenses
Identify the part of the microscope and its function
Ocular Lens
brings the image to focus for viewing with the eye
What are the four solutions used in a gram stain?
Crystal violet
Gram’s Iodine
Ethanol Alcohol
Safranin
What is the significance of Gram Staining?
most important differential staining procedure in micro because most bacteria are either gram – or +.
This technique permits differentiation of four major categories based upon color reaction and shape
Crystal Violet in Gram Staining
•stains the cells in the smear all the same purple colors. (Primary dye)
Gram’s Iodine in Gram Staining
•causes the primary dye to form crystals in the cell walls.
–The peptidoglycan layer in gram + cells are much thicker, causing the entrapment of the primary dye to be far more extensive.
ETOH Function in Gram Staining
dissolves lipids in the outer membrane and removes the dye from the peptidoglycan layer and the gram – cells
Safranin function in Gram Staining
dyes the now colorless, gram – bacteria, but does not affect the gram + because crystal violet dye was not accessible to the ETOH
Steps in Gram Staining
- Heat sterilize loops and slides
- Place one drop of water on slide (small)
- Using loop, smear specimen on slide in water
- Allow slide to air dry
- Heat set slide using flame by passing slide quickly over flame
- Apply Crystal violet stain to slide and allow to soak for 1 minute
- Wash slide with deionized water
- Apply Gram’s iodine to slide and allow to soak for 1 minute
- Wash slide
- Decolorize slide by dropping 95% ETOH on slide and rocking back and forth for 15 seconds
- Flood slide with safranin for 30 seconds
- Wash slide with water and blot dry
Identify the organism, Gram Reaction, Cell Morphology, and Cell Arrangement
Staphylococcus Aureus
Gram Positive
Cocci
Clusters
Identify the Organism, Gram Reaction, Cell Morphology, and Cell arrangement
Bacillus Megaterium
Gram Positive
Bacilli (Rods)
Chains
Spore Staining Technique
- Heat sterilize loops and slides
- Place one drop of water on slide (small)
- Using loop, smear specimen on slide in water
- Allow slide to air dry
- Heat set slide using flame by passing slide quickly over flame
- Cut a piece of blotting paper to place over the smear, just big enough to not hang over the slide.
- Place slide in steam bath and saturate the blotting paper with Malachite green stain for three minutes. Make sure the paper remains saturated
- After 3 minutes, remove the slide from the steam bath and the paper from the slide.
- Thoroughly rinse the slide with a gentle stream of water. The vegetative cells will lose their color during this rinse.
- Flood the smear with safranin for 1 minute. This will stain the vegetative cells.
- Wash the slide and blot dry
After endospores are stained, what color will they be
Green
Acid Fast Stain Technique
- Prepare air dried, heat fixed slide
- Stain slide with carbolfuchsion on blotting paper and steam bath for 5 minutes
- Wash slide with water
- Decolorized slide with acid-alcohol for 15 seconds.
- Saturate smear with methylene blue for 1 minute
- Wash slide with water
- Blot dry
Common Acid-Fast Bacteria and it’s disease state
Mycobacterium tuberculosis
Causes TB (Tuberculosis)
Identify the Stain Technique and the results
Acid-Fast Stain
A) Non-Acid-Fast organism (Blue)
B) Acid-Fast Organisms are Red
Streak Plate (Pure Culture) Technique
- Obtain two different cultures and mix them into one test tube.
- Aseptically smear one quadrant of agar in a dish from the mixed culture tube.
- Flame loop and smear second quadrant of agar after sliding loop through the first quadrant. Continue procedure with the last two quadrants, using the previous quadrant as your source of bacteria
- Incubate the agar plate at 370C for 24-48 hours
Streaked Isolation plate with Isolated colonies in the 4th Quadrant
MSA (Mannitol Salt Agar) is used to isolate which microbe?
Staphylococci
Steps for Isolating Staphylococcus
- Obtain two swabs in sterile solution
- Obtain two Mannitol Salt Agar (MSA) Plates
- Swab inner nose with one swab
- Inoculate MSA plate with nasal swab and incubate at 370C
- Swab an environmental location with second swab
- Inoculate MSA plate with environmental swab and incubate at 370C
What organism will ferment Mannitol? What color will it turn?
•Staphylococcus aureus ferments mannitol and turns it yellow, as the specimen on the left below. Other Staphylococci grow in the MSA, but do not ferment the mannitol.
Identify the Organism
Staphylococcus Aureus
Identify the type of organism
Yeast (Fungi)
Identify the type of organism
Mold (Fungi)
Identify the type of organism
Protozoa
A bubble in the tube for Carbohydrate fermentation means?
Gas was produced when the organism was consuming the carbohydrate
Identify the Carbohydrate Fermentation Results
Starch Digestion
After incubation of a specimen on starch agar, flood the plate with gram’s iodine. If the specimen digested starch, there will be a clear space around it.
Catalase Production Results
If the specimen bubbles when flooded with H2O2, then the specimen is positive for catalase production.
Hydrogen sulfide production Results
•H2S production is indicated in SIM medium by turning the medium dark brown, almost black.
What is Indole Production’s indicator solution
Kovak’s reagent
Indole Production Results
Indole production is indicated by a pink top after 20 drops of Kovacs reagent.
Urea Digestion Results
Certain bacteria produce urease which breaks down the urea into ammonia and carbon dioxide. Inoculating a nutrient broth that contains urea and phenol red indicator will test for urea digestion by urease. If the broth turns pink (left tube), it is positive for Urea digestion
Motility Medium (SIM) Results
Motile organisms will spread out from the line of inoculation while non-motile organisms will only grow along the line
Blood Agar Plates (Sheep’s Blood Agar and Chocolate Agar)
•Blood agar plates will determine hemolytic activity if there are clear spaces around the bacteria.
MSA Mannitol Salt Agar Plate
Mannitol salt agar will determine the bacteria’s tolerance of high saline levels. The bacteria on the left has a high tolerance and digested the mannitol salt
MAC (MacConkey Agar)
•MacConkey agar contains bile salts to inhibit nonenteric bacteria.
TSI (Triple Sugar Iron) Test
Three reactions: butt of tube turns yellow (acid) and slant turns red (alkaline) for glucose fermentation; Butt and slant remain yellow (acid) for sucrose and/or lactose fermentation; No CHO fermentation and the tube remains red (alkaline). Gas production will cause cracks in the agar and hydrogen sulfide production turns the agar black.
Describe this process
•To make a serial dilution of 10-7, obtain three 99mL dilute bottles and one 9.0mL. Transfer 1.0mL of the substance into the 9.0mL bottle, then 1.0mL from the 9.0mL to the first 99mL bottle, then 1.0mL from the first 99mL bottle to the second. Similar to what’s shown below. Remember for every 9mL transfer, you add one dilution power. For every 99mL transfer, you add two.
Identify the organism
Giardia lamblia
Identify the Organism
Trichomonas vaginalis
Identify the Organism
Entamoeba histolytica
Identify the Organism
Plasmodium Vivax
Identify the Organism
Clonorchis sinensis
Identify the Organism
Schistosoma mansoni
Identify the Organism
Balantidium coli
Selective Media
- used to select (isolate) specific groups of bacteria;
- chemical substances in the media inhibit the growth of one type of bacteria while permitting growth of another (MSA, EMB, MacConkey)
Differential Media
- distinguishes among morphologically and biochemically related groups of organisms;
- chemical compounds (following inoculation and incubation) produce a characteristic change in the appearance of bacterial growth and/or the medium surrounding the colonies (MSA, EMB, MacConkey)
Enriched Media
supplemented with highly nutritious materials, such as blood, serum, or yeast extracts, for the cultivation of fastidious organisms
MSA Plate
Mannitol Salt Agar
Both Selective and Differential
Differentiates Staphylococcus species
What is the indicator for the MSA Plate?
Phenol Red
EMB Agar
Eosin Methylene Blue Agar
Both Selective AND Differential
contains the dyes methylene blue and eosin which inhibit Gram (+) bacteria, thus favoring growth of Gram( – )
contains lactose, thus allowing for the distinction between lactose fermenters and nonferments
Used on Enterobacteria (Gram (-) Rods)
MAC Agar Plat
MacConkey’s Agar
A selective and differential medium used to isolate members of the Enterobacteriaceae
Bile salts and crystal violet inhibit growth of G+ organisms (selective)
Contains nutrients, including lactose, as well as bile salts, neutral red and crystal violet
Beta Hemolysis
complete hemolysis. It is characterized by a clear (transparent) zone surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus agalactiae are b-hemolytic
Alpha Hemolysis
Colonies typically are surrounded by a green, opaque zone. Streptococcus pneumoniae and Streptococcus mitis are a-hemolytic
Partial hemolysis
Gamma Hemolysis
There are no notable zones around the colonies
No Hemolysis
SIM Medium
This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar (be motile). SIM is commonly used to differentiate members of Enterobacteriaceae.
How are SIM tubes inoculated
with a single stab to the bottom of the tube
Coagulase Test
Coagulase is an enzyme that clots blood plasma. This test is performed on Gram-positive, catalase positive species to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence factor of S. aureus. The formation of clot around an infection caused by this bacteria likely protects it from phagocytosis. This test differentiates Staphylococcus aureus from other coagulase negative Staphylococcus species.
Citrate Agar
This is a defined medium used to determine if an organism can use citrate as its sole carbon source. It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and CO2. If CO2 is produced, it reacts with components of the medium to produce an alkaline compound (e.g. Na2CO3). The alkaline pH turns the pH indicator (bromthymol blue) from green to blue
Methyl Red Test
This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic, succinic, and formic acids).
Urease Test
This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of urea forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media above 8.4 and the pH indicator, phenol red, turns from yellow to pink.
Durham Tubes are used for?
Determining gas production in a Glucose, lactose, and Sucrose
What is the indicator for a Glucose/Lactose/Sucrose fermentation test?
Phenol Red
Synthetic Media
Media which all ingredients in the mixture are known
Complex Media
All chemicals and substances inside the mixture are not known (SBA, Chocolate Agar)
Antiseptics are used where?
On the outside of the body
Antibiotics are used where?
Inside the body
Disinfectants are used where?
On Hard, non-porus, non-living tissue
Examples of Antiseptics
Listerine
Iodine/Betadine
Examples of Disinfectants
Amphyl
Lysol
Examples of Antibiotics
Oxacillin
Ampicillin
Penicillin
Erythromycin
Genamycin
Sulfa (SXT)
Considerations when administering
appropriateness
Side Effects
Dosage
Exposure Time
Mode of Delivery
Allergic Reactions
Mode of Action (Inhibition mode)
Broad or Narrow- Spectrum
Chemical Nature
Mueller-Hinton Agar is used for what type of test?
commonly used for antibiotic susceptibility testing
Spore
a unit of sexual or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions.
Hyphae
each of the branching filaments that make up the mycelium of a fungus.
Sporangia
a receptacle in which asexual spores are formed
What is the Kirby-Bauer test used for?
uses antibiotic-containing wafers or disks to test whether particular bacteria are susceptible to specific antibiotics
Glucose/Lactose/Sucrose Fermentation test positive/Negative result
Indicator
Required Enzyme
Bubble in the Durham tube, yellow color (Positive)
Purple in the tube, No bubble in the durham tube (Negative)
Bromocresol Purple
(Multiple, Lactase, Sucrase)
Glucose/Lactose/Sucrose Fermentation test negative result
Starch digestion test postitive/Negative result
Indicator
Enzyme Required
Bright colony with glowing background (Positive)
(Negative) Iridescent colony w/ dark background
Gram’s Iodine (Added)
Amylase
starch digestion test negative result
DNA Digestion Test Positve and Negative Result
Indicator
Required Enzyme
Bright Yellow colony (positive)
Blue background w Normal iridescent colony (Negative)
Methyl Green (Already in Plate)
DNAse
Catalase production Positive/Negative results
Indicator
Required Enzyme
Postive-Bubble
Negative-No Bubbles
Hydrogen peroxide (Added)
Catalase
Indole Production test positive/negative result
Indicator
Required Enzyme
Positive- Red Top Layer
Negative- No change/ Yellow color
Kovacs Reagent (added)
Tryptophanase
Hydrogen Sulfide Production test Positive/Negative Result
Indicator
Required Enzyme
Positive-Black, Cloudy tube
Negative- Agar Color/ No change
H2S+ Fe2+
Cysteine Desulfurase
Urea Digestion Test Positive/Negative Result
Indicator
Required Enzyme
Positive-Bright Pink
Negative- Orange/Yellow
Phenol Red
Urease
Why are plates incubated in an inverted position?
So the condensation from the lid of the plate does not contaminate the culture or inhibit growth
Steps of a Simple Stain
Air-Dried, Heat-Fixed Smear (Slide)
Add drops of Methylene Blue or Crystal Violet
Rinse w/ DI Water
Allow slide to air-dry or blot
View bacteria under 1000x
How will the bacterial specimen appear in a simple stain?
They will appear the color of the dye against a white background
Steps of a Negative Stain
Drop ocngo red near one edge of slide
add bacterial specimen to the drop, mix sample
Clean another slide
Angle the slide and smear original slide
Glide 2nd slide over the 1st again
Dry original slide on warner
View under 1000x Magnification
Define Ubiquity
Something that is everywhere or very common all the time
Why is it importantto use the lid of the petri dish as a shield when streaking a plate?
To ensure th culture is not contaminated by microbes in the air
Can you expect to have a pure culture from a body site?
No, because the body has more bacterial cells than human cells and can harbor many different types of microbes
What were thre two fungal species used in Lab
Aspergillus
Penicillium
What are the two types of antimicrobial proteins?
Complement (Nonspecific)
Antibodies (Custom)- target Antigens
General Purpose Media used in Lab
Tryptic Soy Agar
What is Agar?
gelatinous substance obtained from various kinds of red seaweed and used in biological culture media and as a thickener in foods
What is the nutritional mode of Fungi
Chemoheterotrophs
Fungal Cell walls are made of
Chitin
How do we differentiate between the Fungi?
Based on how the sporangia look
How to inoculate a TSI Tube
Stab the agar to the bottom and streak the slant. Leave Cap Open for incubation
Capsules in Bacteria
Large Glycocalyx made of repeating units of polysaccharides and proteins
Capsue Stain procedures
Congo red @ one end of slide
Mix loopful of bacteria into congo red
spread mixture across w/ second slide
Dispose of 2nd slide
air Dry
Rinse w Acid alcohol
Counterstain w Carbolfuschin
Rinse w DI Water
What differnt forms of media are used in Lab?
Broth, Semisolid, Solid, Plates
Define Endospore
A Metabolically inert piece of DNA
Why is it important to use older cultures when performing an endospore stain?
Older cultures tend to be more nutrient depleted, causing sporulation
