LabEx1: TOTAL UP! Flashcards
Gram staining procedures?
- Crystal violet for 1 min (Primary)2.Wash with water 3.iodine for 1 min (Mordant)4. Alcohol 10sec (decolorize)5.wash with water6.Safranin for 30sec (counter stain)7. Wash and blot dry
Spore Stain Procedures?
1.Cover the slide with a blotting paper strip2. flood with malachite green (primary) 60 sec3. put over flame for 4min and do not let it dry, just add more malachite green to keep it soak (mordant)4.remove paper (let slide cool for 1min)5. wash slide with water (decolorizing agent)6.Safranin for 1 min (counter stain)7.wash with water and blot dry
Acid Fast Staining Procedures?
1.cover the slide with a blotting paper strip2. flood with carbolfuschin for 60 sec3. put over flame for 4min and do not let it dry, just add more carbolfuschin to keep it soak (mordant)4.remove paper (let slide cool for 1min)5.Alcohol for 10 sec or until slides is clear (decolorize)6.wash the slide7.methylene blue for 1min (counter stain)8. wash with water and blot dry
Streak plate procedures?
- Draw 4 quadrant on the streak plate2. sterilize the loop3. get 1 loop full from the mixed culture4. streak zigzag from quadrant 1 to 45. sterilize loop for each quadrant but dont loop again for 2 to 46. DO NOT CUT ACROSS NO MORE THAN 2 TIMES
EMB plate procedures
- Draw 4 quadrant on the EMB plate2. sterilize the loop3. get 1 loop full from the mixed culture4. streak zigzag from quadrant 1 to 45. NO NEED TO sterilize loop for each quadrant6. DO NOT CUT ACROSS NO MORE THAN 2 TIMES
Smearing procedures?
- sterilized the slides and loop2. transfer few loopfuls of sterile water3.sterilized the loop again4. carefully loop the bacteria from the tube or plate5.smear the loop of bacteria on the slide with the sterilize water.6.air dry by pass the slide through the flame 2 to 3 times
what is resolution? list the factors that affects resolution.
shorter the wavelength of light used, higher resolution.-white light used in compound microscope limits resolution of structures smaller then 0.2mm
how higher magnification affects resolution? how it can be corrected?
higher magnification inverse relationship to lower resolution.
differentiate between virtual and real image.
the objective lens magnifies the specimen to produce real image that is projected to ocular lens to produce the virtual image.
list 4 points regarding proper use and storage of microscopes.
- use both hands when you carry a microscope to your table.2.plug in the lamp cord into electrical outlet, then turn on the main switch of microscope3. before storing microscope back, replace the low objective lens into working position4. always check before change objective lens to avoid slide breakage.
identify at least 4 differences between prokaryotes and eukaryotes.
prokarytoes:-non membranous-one circular chromosome-divided by binary fission-made of peptidoglycanEukaryotes:-separated from cytoplasm-both membranous & non-membranous-no peptidoglycan-divided by mitosis
what is the biochemical basis of cheek cell staining process, are the cheek cells prokaryotes or eukaryotes?
methylene blue is used to stain animal/human cells to make nuclei more visible under a microscope.-the cheek cell is a eukaryote, because eukaryotic cell contain a nucleus.
Staining:define primary stain
mainly stain all cells
Staining:define mordant
holds the dye down
Staining:define decolorizing
wash of primary stain
Staining:counterstain
color to cells that dont retain the primary stain
describe the principle of gram’s staining process, include the role of each chemical reagent used in the staining process.
To distinquish between two cell wall gram positive and negative.
advantages of the gram’s staining process
advantage:-it’s a relatively rapid and an extremely useful tool-the staining provides suggestive includes during the identification process-it provide valuable information so as to initiate course of antimicrobial treatment
disadvantages of the gram’s staining process
disadvantage:-often, disease causing bacteria, does not have distinct stain characteristic -the stain is not specific enough to diagnose the cause of most infection-some bacterial cells stain poorly or not at all
list 3 precaution for the gram’s stain
1.fixing2.staining3.timing
identify the 2 bacteria’s which characteristically produce spores. name the bacteria you did for that experiment.
1.spore has a thick wall2.is a metabolically inactive structureBacillus anthrasis or Clostridium tetanus
what is 5 features of spores and list spore position inside vegetative cells
- spore begin to isolate DNA replicate2. plasma membrane surround DNA3. spore septum surrounds isolated portion forming forespore4.peptidoglycan layer forms between membrane5.spore coat forms then endospore freed from cell
what are functions of spore?
are reproductive cells that must fuse in pairs in order to give rise to a new individual.
what are differentiate between spore and vegetative cells?
- endospores is form from vegetative cell2.vegetative cell response to environment signals
what is sporulation and germination?
1.sporulation- process of spore formation in a sequential event2.germination - bacterial spore transform into a vegetative cell upon return of favorable conditions by the metabolic process
name 2 bacteria’s which are acid fast positive and the associated disease they cause in humans.
Mycobacterium nocardia>Mycobacterium tuberculosis - causes chronic diseasesMycobacterium leprae - hansen’s diseases
describe the composition of the cell wall that is responsible for the acid fast properties of the cell.
Acid Fast Bacteria are Gram positve, peptidoglycan contain large amout of lipids (mycolic acid).
What is sterile transfer technique?
they incorporate procedures that minimizes chance of other organisms being accidentally introduced.
list 4 factors with regard to importance of the sterile transfer techniques.
- free from containing you or others2. prevent culture from contaminating you or others3.to isolate pure culture of microbes not considered4.prevents observing features of microbes
name 2 methods for pure culture isolation.
1.spreading plating on solid agar medium with a glass spreader.2.streaking plating with a loop
list 3 common errors in colony isolation by the streak process
1.label all media, tubes and cultures2.use a loop which is sufficiently cooled3. avoid aerosols4. disinfects work place
why pour plate technique needs to be done at a faster pace compared to the streak plate method
pour plate needs to be cooled enough before it solidify while streak plate doesn’t
what is peritrichous arrangement of flagella?
peritrichous - Multiple flagella may be randomly distributed over the entire bacterial cell
what is amphitrichous arrangement of flagella?
amphitrichous - A single flagellum (or multiple flagella; see below) can extend from both ends of the cell
what are spirochete?
are flexible, is a member of the phylum Spirochaetes, which contains distinctive diderm bacteria, most of which have long, helically coiled cells
name the disease that caused by the spirochete Borrelia burgdorferri
predominant causative agent of Lyme disease
Acid Fast Positive -
Clumped together “cord factor” virulence factor (red in color due to carbolfuschin)
Non-Acid Fast (negative) -
blue in color due to methylene blue
Emb plate
EMB contains dyes that are toxic to gram-positive bacteria. EMB is the selective and differential medium for coliforms. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1. A common application of this stain is in the preparation of EMB agar, a differential microbiological medium, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose
emb plate contain
lactose sugar, peptones, sodium deoxycholate, eosin, methylene blue, extracted best yeast
1.Carrying a microscope to your table from the storage cabinet, you should
Use both handsDON’T- disassemble any part of the microscope without permission from the instructorDON’T - apply force on any part of the microscope
2.before and following microscope use, you should
clean all lenses with lens tissue paperDON’T - use paper towel or kleenex, as they may scratch the lends
3.before you turn on the main switch of the microscope, you should
plug lamp cord into the electrical outlet first.
- place slide in the _______
slide holder
5.raise the stage as close to the 4x objective as possible using the ___________.
coarse adjustment knob (on the sides)
- while viewing through eyepiece, you should
adjust the stage downward, so that the slide and lens move apart from each other
- To maintain sharpen focus with the fine focusing adjustment knob, you should
use one hand to manipulate the specimen slide and other hand to turn the fine focusing knob
8.before storing microscope back, you should
replace the low objective lens into working position.-the microscope is always stored with the lowest power objective lens locked in.
- place the ________ to avoid dust on the microscope
plastic cover
10.always check before changing objective lens to avoid ________
slide breakage
-Advantages: easy to use, readily and relatively inexpensive, commonly used-Disadvantage: can recognize cells but not finer details; cells lack contrast with water -Contrast:Difference in intensity between objects and it’s background
Light or bright field microscope
100x objective lens requires ?
Oil immersion
What does oil immersion do the lens?
-Oil displaces air, avoids refraction-oil increases numerical aperture, thus improves resolution because more light rays are gathered into lens to produce image.
___________ is bending of lights ray as it passes from immediately to another
Refraction
Refraction prevents
Light from entering the small opening of the high powered objective lens
Aperture size of the objective lens decreases with increased magnification; which allows?
Less light to enter the objective lens
-contrast special condenser to enhance contrast when viewing object-permits examination of internal structures -good for viewing objects without staining (as cilia and flagella)
Phase contrast
-objects look bright against dark background -contains special condenser with opaque disc to block light-good for viewing motility of organisms and examining microbes that Cannot be stained or they get distorted by staining procedures
Dark field
-normally use to visualize organisms that fluoresce-often cells are treated with fluorescent dyes then observed for fluorescence
fluorescence microscope
-uses electromagnetic lenses, electron and fluorscent screen to observe specimen instead of glass and light-wavelength of electrons is approximately 1000x shorter than visible light-much higher resolution is obtained and greater detail can be observed
electron microscope
_____ technique of adding dyes to color specimen (microbes, cell, tissue etc)Functions: provides contrast and increases resolution for light microscope-allows visualization with ease-reveals internal and external structures-aids in classification and identification
STAINING
_______ thin film of material containing biological material or microorganism spread over the surface of slide.importance of _________________ thick - trouble seeing individual cells_________ thin - you may find no organismtoo much stirring - disrupts cell arrangement
SMEAR
________ adherence of microorganism to slide-accomplished by either heat or chemicals (cold alcohol acetone mixture)-advantages: ______ inactivates the microorganisms-prevents specimen slipping in subsequent stain application and washing steps-makes subtle alterations so that organisms more readily accepts stains
FIXING
________ an additive added to dye solution, to intensify the stain, it binds to the dye and makes it less soluble-it increases affinity of stain for biological specimen, stain is retained better-coats certain structures, so that it’s easier to visualize following staining.
MORDANT
_____ involves application of a single dye to a smear-highlights entire organism; shape and basic structure’s are visible, including arrangement of cells-are of relatively less value in diagnostic bacteriology
Simple Stain
_____ involves application of two or more dyes together or separately to a smear._mainly used to distinguish one group of bacteria from another-various dyes reacts with different bacterial structural components
Differential Stain
types of differential stain
Gram StainAcid Fast StainSpore Stain
3 types of special stains that are used to color and isolate specific parts of microbes
Negative StainFlagella StainFluorescent Stain
______ gives an idea of cells shape + size-presence or absent of capsule can be detected
Negative Stain
What type of dye you use for cheek cells(eukaryote)?
Methylene blue (basic dye)
Steps of locating microbes under 100x magnification
- locate microbe with 10x2. lower the stage3. place small drop of immersion oil on the cover slip
