Eating Habits - Lab Flashcards
Don’t place dirty slide in this beaker / tray.
cleaning solution
discarding contaminated materials
Remove all labels from tubes, placing them in appropriate racks on side of the room as shown
petri dishes with cultures
and contaminated swabs go in the biohazard bags
Dispose of used glass pipettes
in the discard pans on the side desk or placed on your desk. At the end before you leave lab, transfer all pipettes in one pan.
Don’t discard bulbs and pumps
in the pan
Before storing microscope back
replace the low objective lens into working position. The microscope is always stored with the lowest power objective lens locked in. Place plastic cover to avoid dust on the microscope
Condenser
focuses light on the specimen
- is a lens system located below the stage
- it doesn’t affect magnification
- may include mirrors and prism to deflect light
Iris diaphragm lever
adjust light intensity for best contrast
- is part of the condenser system, below the stage
Coarse knob
Coarse knob: allows for gross focusing
Fine focus knob
Fine focus knob: allows for fine tuning of the focus
Head
Head: supports the objective and ocular lens system
Ocular lens
lens in each eyepiece; lens closest to the eyes
provides additional 10 x magnification when used with objective lens
monocular vs binocular microscopes (?)
Objective lens
adjustable lenses on the revolving nosepiece
normally used to change magnification of a specimen
built to minimize distortion in specimen image / color
4 types of objective lenses on microscope (scanning, low, high and oil immersion)
Total magnification
times 10
Resolving power - aka resolution - (2)
refers to ability of lens to distinguish between 2 points, a specified distance apart
Resolution is dependent on
quality, specimen prep, numerical, wavelength, white light
quality and type of lenses the magnification
how the specimen has been prepared
numerical aperture of lens (i.e. ability of the lens to gather light)
- larger the numerical aperture, better resolution
wavelength (?) of the light source; can limit resolution at higher magnification
shorter the wavelength of light used, higher the resolution
white light used in compound microscope limits resolution of structures smaller than 0.2μm
Working Distance (your objective)
refers to the space between slide specimen and objective lens - higher the magnification, shorter is working distance
Usefulness of a microscope depends on degree…
of magnification and resolve
Light or bright field microscope
easy to use, can recognize cells but not finer details. cells lack contrast with water.
Contrast
difference in intensity between object and it’s background
100x objective lens - how it works (drown)
- oil displaces air, avoids refraction - oil increases numerical aperture (?), thus improves resolution
because more light rays are gathered into lens to produce image
Refraction
prevents light from entering the small opening of the high powered objective lens
Refractive index
is measure of the velocity of light as it passes through medium
Phase contrast
- contains special condenser to enhance contrast when viewing object - permits examination of internal structures- good for viewing objects without staining (as cilia and flagella)
Dark field
objects looks 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
Fluorescence microscope
normally use to visualize organisms that fluoresce
- often cells are treated with fluorescent dyes then observed for fluorescence
Electron microscope
uses electromagnetic lenses (?), electron and fluorscent screen to observe specimen instead of glass lenses and lightwavelength of electrons is approximately 1000x shorter than visible light
much higher resolution is obtained and greater detail can be observed.
Drawbacks to electron microscope
Lenses and processed specimen must be enclosed in a vacuum unit (air absorbs electron, expensive. Processing increases artifact chances and cannot be used to study living organisms
staining
contrast and light, internal and external structures, identification
provides contrast and increases resolution for light microscope- allows visualization with ease- reveals internal and external structures- aids in classification and identification
smear
smear thick - trouble seeing individual cells smear thin - you may find no organism too much stirring - disrupts cell arrangement
fixing (pipes)
adherence of microorganism to slide - accomplished by either heat or chemicals ( cold alcohol acetone mixture)
advantages of fixing
fixing inactivates the microorganisms- preserves major parts with minimum distortion - prevents specimen slipping in subsequent stain application and washing steps - makes subtle alterations so that organisms more readily accepts stains
mordant - purpose and what its made of
Fixes the dye. Iodine. an additive added to dye solution, to intensify the stain- it binds to the dye and makes it less soluble
advantages of mordant
like, coat, see
- it increases affinity of stain for biological specimen, stain is retained better- coats certain structures, so that it’s easier to visualize following staining
stains are made of what substance..
salts composed of positive and negative ion; only one part is colored. Chromophore: colored part of a stain
Basic (cationic) dye
positive part is colored - methalyne blue, crystal violet, malachite green
Acidic (anionic) dye (travel)
negative part is colored, sudan red, india ink
Simple stain
involves application of a single dye to a smear. highlights entire organism; shape and basic structures are visible, including arrangement of cells - are of relatively less value in diagnostic bacteriology
Differential 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
Special stains - types FFN
FFN
3 types of special stains:
A. Negative stain (example: capsule stain) B. Flagellar stain C. Fluorescent stain
3 types of differential stain
gram, acid fast and spore stain
special stain
dye or color isolates specific structure of microbe.
negative stain uses what type of dye
on bacteria, uses anionic dyes. - chloroform.
bacteria is repelled by
acid. remains unstained.
halo around cell is…
negative acidic, likely the capsule
advantages of negative
get an idea of size and shape, and evidence of the capsule
what part gets stained in basic stain
the cell itself gets stained
chromatophores
part of plasma membrane. light reflecting cells.
euglena
looks like snap peas, has flagella. algae, eukaryote. bright green.
paramecium
dirty brown, eukaryote
stains (made of)
dyes, salt positive and negative ion
center of cheek stain dark part is
nucleus DNA
chromophore
positive part, blue part attracted to negative charge, DNA.
DNA negative part gets dyed due to
phosphate groups
gram negative color
pink and red
gram positive stains..(color)
peptioglycan, purple
decolorizing (nail polish +)
Alcohol/acetone. critical step. washes off stain. gram positive, water sucked out.
spore stain is a __stain
differential stain - can be positive or gram negative
gram positive rods..(ex)
make spores. only clostridium and bacillus anthracis.
use oil immersion lens with..
gram stain. no coverslip needed
acid-fast staining steps (car steam)
you’re mixing so it’s called mixed stain. primary dye - carbolfuscia dye on top of paper towel. heat with paper towel is fixing - steam acts as mordant. keep adding more carbonfuscia as you heat it. counterstain methalyne blue
real and virtual image
objective to real to ocular, ocular to virtual
The objective lens magnifies the specimen to produce real image that is projected to ocular lens . The real image is magnified by ocular lens to produce the virtual image.
virtual image is what position
upside down
stain used for cheek cell
alwasys simple stain, one dye
blue dye disassociates (eyes)
negative and positive. chromatophore is positive part, dye is attracted to negative part.
cheek cell is
eukaryotic cell
dark dots and rod shapes in cheek stain
are bacteria - prokaryotes
endspore stain is what type of stain
differential stain
advantages of gram staining
fast - gonnorhea, clues - strep pneumonia, treatment
it’s a fast and useful tool ex: urethral secretion (neissan gonorhea - only gram - diplococci) provides clues during the identification (lungs - strep pneumonia) for antimicrobial treatment (gram - like syphillis and TB can’t be treated)
disadvantages of gram staining
disease not distinct, can’t identify infection, stain poorly, need new cells
often, disease causing bacteria do not have distinct stain characteristics - the stain is not specific enough to diagnose the cause of most infection - some bacterial cells stain poorly or not at all; freshly grown bacteria is preferred. aged or old cells = misleading results
endspore staining process
primary stain - malachite green (basic dye). mordant = steaming (softens keratinized spore coat). wash and add secondary stain = safranin (basic dye, counter stain). Steaming needs to be done properly or it will be a light stain.
endospore - green is the..
spore released from mother cell
red endospores
mother spores (vegetative cells)
dye that can’t enter mother cells
malachite green
endospore staining bacillus anthracis
spores and vegetative cells. bacillus gram stain will always look like glass beads.
spores only made by gram..
positive
acid fast stain used because..
it’s another differential staining used for bacteria which do not readily stain by the Gram’s reaction although they belong to Gram positive group
bacteria staining in acid fast (acidic beans)
Staining is facilitated by heat and phenol (present in the primary stain). Once stained, these cells are able to retain the stain because of the waxy lipid mycolic acid cell wall that are not easily decolorized by acid alcohol, so they appear red
non-acid fast cells in acid fast stain
non acid fast cells easily decolorize and can be counterstained with another dye (methylene blue) and they appear blue
acid fast procedure (blue car oil)
primary stain carbolfuschin. Apply mordant, heat and phenol (from the primary stain) by gently heating. use paper towel. STEAM IS THE MORDANT. decolorizer is acid alcohol. counterstain methylene blue. observe using oil immersion lens
Acid fast organisms - which colors stain what
acid fast is stains red; non acid fast organisms stains blue
non acid fast organisms (me)
stains blue
what type of staining is acid fast?
differential staining
we used _ bacteria in acid fast
gram +. don’t stain with gram stains, due to mycolic acid.
in acid fast cells w/ mycolic acid, (car)
retain the carbolfuscion
cord factor in acid fast
helps the pathogen to escape from host defense and being destroyed. it is a virulent factor.
cord factor is made of
secreted protein and it sticks cells together. individually shaped cells are rod shaped.
2 important bacteria used in acid fast in lab
m. tuberculosis and hansen’s disease (leprosy) called micobacterium leprae. Mycobacterium and Nocardia sps.
cells w/ mycolic acid (acid fast) (1st car)
will retain primary stain, carbolfuschion
no mycolic acid - acid fast stains what color
blue
identifies TB
acid fast
importance of sterile transfer techniques (newport)
not contaminated, protect me, isolate culture
keep bacterial specimen being studied, free of contaminants from environment; this is critical when a pure culture is desired
to prevent culture from contaminating you or others; this ensures to help yourself and others safe
to isolate pure culture of microbes
prevents observing features of microbes not considered as part of your experiment (contaminants are kept out); enables us to study metabolism of a particular organism
precautions during sterile transfer
label, cool loop, no aerosols, high above flame, gently dry, don’t touch anything after sterile, Disinfect, no books
Carefully label all media, tubes and culture
Use a loop which is sufficiently cooled; don’t wave to cool
Avoid aerosols: loop has heavy load of residual microbes remaining
- hold loop high above flame
- gently dry it, before dropping to red hot area of flame
Keep transfer implements sterile
- do not touch any surface with it once flame sterilized
Disinfect work area before and after, remove unnecessary books and papers
petri plates are labeled on
the bottom in case they are dropped and lid comes off
petri dishes are placed in the incubator…
upside down due to water evaporation
streak plate
widely used to obtain single colonies, which represents a pure culture of the bacterial cells.
cultures used in streak plate (selene)
s. aureus and Serratia marcescenes
common errors w/ streak plate (folsom)
fresh cells, pass twice, edge to edge, loop verticle, shake tube
Getting fresh cells on loop (from original culture tube) before streaking the 2nd and 3rd section
2. Failure to pass through previous section two times
3. Failure to go from edge to edge in each quadrant
4. Holding loop vertically; it’ll gauge agar
5. Forgetting to shake the tube, when obtaining cells for streaking in the first quadrant.
differences in streak/spread plate and pour plate
spread plate - separated colonies are on agar surface and use inoculating loop to pick up cells from isolated colonies. pour plate - separated colonies are in between agar layer and use inoculating needle to stab agar and pick up cells. don’t dig.
why are some colony sizes smaller when cells are closer? (SNW)
SNW
Due to competition for space, nutrients and metabolic waste accumulation that could be inhibitory to other bacteria
emb agar - carbon source
lactose sugar, fermented, generates acid.
emb agar - peptones
hydrolyzed proteins, source of nitrogen and amino acids
emb agar - eosin dye is the…
differential factor
emb agar - methlyne blue - which factor
selective factor, only supports growth of gram - rods.
emb agar - extract
provides vitamins and essential nutrients
emb agar process
for isolation, detection and confirmation of intestinal bacteria.
bacteria used in emb
e.coli and enterobacteria.
green sheen in emb
e.coli. eosin precipitates on colonies under acidic medium due to lactose fermentation. stable acids made, ph lowers. eosin dye precipitates turns green
red emb
enterobacteria, eosin dye doesn’t precipitate bc lactose sugar ferments and forms unstable acids. fish eye colonies.
Biochemical basis of selectivity and differential ability of EMB media (analise-no positive)
Eosin Y and Methylene Blue is differential and selective
selective because Eosin methylene blue agar contains aniline dyes that don’t allow growth of gram positive. Large amounts of acid from outside fermenters. Cause dye to precipitate on the colony surface producing a green color and pink.
why is emb a complex media?
Because it uses different dyes to rule out different bacteria growth. This makes it a complex media because it can do several things at once.
mordant in gram stain is
Iodine is as a mordant so dye can’t be removed easily
decolorizing in gram stain (gina)
closes the pores in the cell wall and prevents the stain from exiting the cell.
mordant in acid fast (melt)
heat, allows the primary stain to penetrate the waxy mycolic acid layer. The heat will prevent the cells from being destained using acid-alcohol
decolorizer in acid fast does what (clean)
strips the stain in non acid fast cells. won’t enter the cell wall of acid-fast bacteria
fixing in endospore stain
penetrates endospore, dye goes into peptidoglycan. heat is the mordant.
primary stain in endospore stain driven into cells with…
heat
counterstain in endospore stain binds to (poly)
lipopolysaccharide layer - gram -
treponema palladium
(syphilis) doesn’t stain by gram stain or acid fast
green sheen on agar plate is always..
e. coli
oil immersion use
40X objective, focus and center the object
Lower stage and put a drop of oil on the slide
Change to 100X objective
Raise the stage using the coarse adjustment knob until objective lens touches the slide and the immersion oil on the specimen
Look through the eyepieces and lower the stage slowly, using the coarse adjustment knob
using the coarse knob until you see the specimen and then use the fine focusing knob
You may need to adjust using the fine focusing knob.
resolution at high magnification
increase light intensity
Aperture size of the objective lens decreases with increased magnification; this allows less light to enter the objective lens. In such situation, with high magnification you may need to increase light intensity to obtain a good image of the specimen
capsule stain - negative stain
distinguishes capsule from bacterial cell. The capsule stain uses acidic stain and a basic stain to detect capsule production
advantages of negative stain
bacteria are not heat fixed so they don’t shrink, and some bacterial species resist basic stains (Mycobacterium) and one way they can be visualized is with the negative stain
what happens in gram positive in gram staining
cell wall collapses and crystal violet trapped
starch hydrolysis - exoenzymes
excreted out of cell, they break down extracellular substances, ex. amalyse.
starch hydrolysis - endoenzymes
are enzymes used inside metabolic reactions. ex. endoenzyme ex. glycolic pathway and catalase.
starch hydrolysis - process (potatoes)
use iodine to detect intact hydrolysis. iodine + intact starch = dark colored (blue). amalyse negative. iodine starch hydrolysized = halo around colonies. means it’s amalyse positive (cells secrete amalyse).
starch hydrolysis detects which enzyme
a-amylase
catalyse
Catalase is an enzyme produced by many microbes to neutralize the toxic effect of hydrogen peroxide. It breaks down hydrogen peroxide to generate water and oxygen. Oxygen release from the peroxide molecule is observed as bubble. Catalase presence can be detected by adding hydrogen peroxide directly onto bacterial culture tube or alternatively onto a dry smear of bacterial cell transferred to a slide
results of catalyse and peroxidase
if you don’t see bubbles, absence of catalyst and peroxidase
bubbles in catalyse
catalyse positive
catalyse enzyme is a
endoenzyme
what is generated in gas pak
h20 gas and CO2 generated inside chamber. can be noticed as moisture inside.
anaerobic growth in gas pak
due to existing spores
anaerobic bacterial growth can be cultivated (thyroid)
thiogycellate medium (absorbs dissolved 02) makes it unavailable for bacteria. physical method of heating followed by cooling, inoculating and tight screwing of cap, aneorobic jar w/ gas pak
tsi - entobacterium - color change due to..
acid formation
tsi - e.coli
use all 3 sugars. (slant) growth (butt) acid and gas. acid and gas production. yellow from fermentation and increases the acid ph. medium has been pushed up because gas was generated during fermentation.
tsi - enterobacteria (instestines)
use all 3 sugars. (slant) growth (butt) acid and gas. intestinal bacteria as well, uses glucose first, then shift to use lactose and sucrose and it fermented, producing acid and gas.The medium has been pushed up because gas was generated during fermentation.
tsi - proteus
(slant) growth (butt) growth. it uses glucose, but it cannot use lactose or sucrose. So it used amino acids of the peptones, the ammonia released and forms ammonium hydroxide. The blackening is due to anaerobic respiration and iron sulfide.
blue on endospore slide is
endospore
red on endospore slide is
bacillus anthracis - vegetative cell
capsule stain is done using___stain
negative
emb test uses what types of medium?
selective and differential medium
(ex of selective media) thayer martin media used for
the isolation of neissan gonhorrea. gram -. This is selective medium.
(ex of selective media) sabaroud’s is for..
fungi. it contains low ph, high salt and sugar. - inhibitory
emb stands for
eosin methalyne blue
emb uses what method?
streak plate
in starch hydrolysis test, we are looking for..
amalyse. Amalyse breaks down starch to glucose units, then glucose is absorbed by bacterial cells.
incubate plate at 37 degrees C
emb
after you heat EMB, you use
iodine to detect intact or hydrolized starch. iodine reacts with intact starch to form dark color (washing machine). if dark, no amalyse.
gram stain - primary and counterstain and mordant, decolorize
primary - crystal violet, counter - safranin, mordant - iodine, Decolorize - alcohol: acetone
acid-fast - primary and counterstain and mordant
primary - carbolfuscion, counter - methalyne blue, mordant - heat. decolorize - acid alcohol
spore stain - primary and counterstain and mordant
primary - malachite green, counter - safranin, mordant - heat. no decolorizer, primary stain washes off with water
gram stain principles
differential stain, violet and iodine enters both + and -, forms crystal and iodine complex, alcohol removes stain from gram - only. alcohol dehydrates and removes h20 molecule. peptido in gram + collapses and traps iodine crystal violet complex. gram - outer membrane is destroyed and crystal violet comes out of cell. alcohol really important.
eosin is an ___dye
acidic
tsi - pseudomonas (ramona)
it is an aerobic bacteria, so there is no growth in the butt. It uses glucose first, it cannot use sucrose or lactose, so it uses the peptone amino acids as a carbon source. The blackening is caused by small amino acids, sulfur amino acids can be cysteine or methionine. They get diaminated, amine group released as ammonia, combines with water in medium to form ammonium hydroxide, it trickles down medium turning it red.
peritrichous
e.coli
monotrichous (ramona)
pseudomonas
ampitrichous (voltans)
spirillum volutans
deep butt
creates anaerobic, no oxygen. only anaerobic can thrive (or fermenting)
shallow slant
provides aerobic
purpose of TSI
to determine enterics from non enterics
tsi - glucose is the lowest..
concentration, usually preferred energy source
fermentation (alcohol)
pyruvic acid becomes either alcohol or lactic acid, then becomes acidic, few atp made
respiratory
glucose becomes pyruvic acid, then goes to kreb. pathway can be areobic or anaerobic
sodium thiosulfate in TSI is (runner up)
alternate electron acceptor in aerobic respiration, then it becomes hydrogen sulfide. then combines w/ iron sulfate and becomes iron sulfide. blackening in butt.
strawberry red due to
ph indicator, alkaline acid. high ph will turn it red.
carbon source in TSI
sugar
yellow is tsi
is from fermented sugar
iron sulfate detects (hs)
hydrogen sulfide
iron sulfide is what color?
black
pseudomonas - respiration is…
aerobic bacteria
black in pseudomonas (sis)
due to cysteine and methionine amino acids
acidic
low ph
alkaline
high ph
gas in e coli due to
fermentation. both e coli and enterobacteria have growth in butt.
