Final Set - Lab Final

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Simple stain

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

Negative stain

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

Differential stain

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

Basic stain

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

Acidic stain

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

Gram stain

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

Capsule stain

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

Endospore stain

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

Starch hydrolysis

The above test is used to determine what?

Starch agar

What type of agar is used in this test?

Positive

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

Flagella stain

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

Sugar fermentation test

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

Glucose, lactose and sucrose

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

gas production, Durham Tube

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

Phenol Red

What indicator is used in the above sugar fermentation test?

The first yellow tube (A/-)

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

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

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

The pink tube (K)

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

The second yellow tube (A/G)

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

Motility

The above test is used to determine what?

The first and last tubes

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

Motility test medium

What type of agar is used in the above test?

Catalase test

The above image is of what test?

The sample of the right

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

H2O2, hydrogen peroxide

What reagent is used in the above test?

The organism grown on the plate produces the enzyme catalase

What does this test determine?

Oxidase test

The above image shows what test?

Positive

Would this be a positive or negative oxidase test?

Chromogenic reducing agent

What reagent is used in the above test?

Nitrate reduction test

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

Tube 2

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

Tube 3

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

Urease test

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

Phenol red

What is the indicator used in the urease test?

The pink tube (+)

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

Both the yellow and orange tubes (-)

Which one of these tubes is negative for urease hydrolysis?

Antibiotic resistance and susceptibility

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

Mueller Hinton

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

The zone of inhibition

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

Staph aureus

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

Mannitol fermentation and the reduction of the pH

What caused section 1 of this plate to turn yellow?

Gram-negative organisms

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

Staphylococcus

What organism does the above medium favor the growth of?

Coagulase test

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

The bottom tube

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

To differentiate S. aureus from other gram-positive cocci

Why would you run a coagulase test?

CAMP

The above image is an example of what test?

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

What does the CAMP determine?

Beta hemolysis

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

Alpha hemolysis

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

Gamma hemolysis

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

Bacitracin

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

Beta, streptococci, beta

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

Blood agar

The above bacitracin susceptibility test is run on what medium?

Bile esculin

The above test is grown on what medium?

Positive, any darkening of the medium is considered positive

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

Enterococci, Streptococcus bovis

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

MacConkey agar

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

Neutral red

What is the indicator in MacConkey agar?

Gram-negative

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

Ferment lactose

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

Coliform

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

EMB, eosin methylene blue

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

Gram-negative

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

Coliform

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

Sulfer production, indol, moltility

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

black precipitate

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

Red band on top of medium

What would a SIM tube positive for indole look like?

Entamoeba histolytica

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

Giardia lamblia

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

Trichomonas vaginalis

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

Balantidium coli

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

Plasmodium vivax

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

Plasmodium falciparum

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

Saccharomyces cerevisiae

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

Penicillium notatum

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

Aspergillus niger

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

Ascaris lumbricoides

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

Ascaris lumbricoides

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

Enterobius vermicularis, pinworm

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

Enterobius vermicularis, pinworm

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

Fasciola hepatica

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

Schistosoma mansoni

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

Schistosoma mansoni

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

Schistosoma japonicum

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

Taenia solium

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

Taenia solium

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

Taenia solium

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

Serratia marcescens

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

Miccrococcus luteus

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

Microscope-Ocular Lenses

Top of microscope (binocular) magnifies 10x

Microscope- Objective Lenses

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

Microscope- nosepiece

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

Microscope- parfocal

ability to maintain focus when you change to a higher magnification

Microscope-arm

supports ocular and objective lenses

Microscope-base

supports entire microscope

Microscope-stage and mechanical stage control

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

Microscope-condenser with condenser lens

aims light through the specimen

Microscope-substage adjustment knob

silver left side, this is for the condenser

Microscope-diaphram

regulates the amount of light passing through the condenser

Microscope-Coarse Adjustment Knob

allows for focusing

Microscope-Fine Adjustment Knob

allows for focusing- fine tunes focus

Microscope-Lamp Control Knob

round button at the bottom regulates the amount of light used

Microscope Inversion

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

Total Magnification

Ocular lens mag X objective lens mag

Resolution

ability to distinguish 2 separate points- makes it clear

Contrast

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

Diameter of Field

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

Darkfield Microscope

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

Phase Contrast Microscopes

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

Fluorescence Microscopes

Uses ultraviolet light source and fluorescent stains (called Fluorochromes)

Flurochromes

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

Immuneofluorescence (Fluorescent antibody staining)

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

Electron Microscopes

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

Transmission Electron Microscopes

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

Scanning Electron Microscopes

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

Scanning/tunneling; Atomic Force

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

Calculating diameter of field

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

diameter ratio equation

LPD = SPM
SPD LPM

5 ways to help with resolution

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

Standard smear

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

2 reason to heat fix slides

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

2 type of staining

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

Acidic Stains

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

Basic Stains

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

Simple staining

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

Negative Staining

Use 1 acidic stain (colored portion is negatively charged) to color the background around cells. Allows visualization of size, shape, arrangement and number.
Drop of Nigrosine at end of slide
put Bacillus Magaterium in drop
spread with new slide
do NOT heat fix, just air dry because you are not adding a stain that has to be rinsed.

Capsule stain (nic)

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

Differential Staining

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

What is Gram Staining

Steps to Gram Staining (cges)

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

What is acid fast staining

Steps to Acid Fast staining (hcab)

Standard smear of Staphylococcus Epidermidis and Mycobacterium Smegmatic, air dry and heat fix
On hot plate place slide and paper towel over weigh boat and heat while staining with CARBOLFUCHSIN (4-5 min., then cool and rinse
decolorize with ACID-ALCOHOL 3-5 sec.
Counter stain with METHYLENE BLUE 30 sec.
End up with Red-(acid-fast)mycobacterium and Blue Staph

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

Mycobacterium and Nocardia

Endospore Stain

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

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

Bacillus Magaterium

Bacteria used in acid-fast and gram staining

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

List 3 factors with influence clinical specimen quality

Proper collection, labeling, and Transport

Potential problem is specimen is not handled carefully

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

Proper Specimen collection for blood, throat swab and sputum.

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

Proper specimen collection for wound, fecal and Gonococcal Specimens

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

2 types of Antimicrobial agents and description
and other Antimicrobial agents

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

antibiotic that inhibit cell wall synthesis

Penicillin

antibiotic that inhibits protein synthesis

Tetracyclines

antibiotic that disrupts cell membrane function

Anti-fungal Drugs (zoles)

antibiotic that inhibit Nucleic Acid Synthesis

Cipro

antibiotic that interfere with enzyme function

sulfa drugs

What is the Kirby-Bauer Test for Antimicrobial Sensitivity

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

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