Micro lab quiz 3
Which of the following are the expected results of staining bacteria with Malachite Green followed by counterstaining with Safranin?
Vegetative cells will appear pinkish-red, while any endospores present will appear green
The purpose of serial dilution and plating
Reliably quantifies bacterial load and isolate individual colonies
Define the sizes, morphologies (shapes), and arrangements that bacterial cells come in
SHAPES: cocci - (singular: coccus) spheres Bacilli - (singular: bacillus) rod-shaped Vibrios - slightly curved rods Coccobacilli - short rods Spirilla (singular: spirillum) AND spirochetes - spiral-shaped Pleomorphic - multiple shapes ARRANGEMENTS: Diplo- - cells arrange as pairs (ie diplocbacillus or diplococcus) Staphylo- - cells arrange as grape like clusters (ie staphylococcus) Strepto- - cells arrange in chains (ie streptococcus or streptobacillus) Tetrad - a quartet of cells Sarcina - two quarters of cells that form a 3D cube
Gram-positive bacteria
They have cell walls with a thick layers of peptidoglycan (peptides that cross-link sugar chains) and with the addition of alcohol the peptidoglycan becomes dehydrated, trapping the crystal violet iodine complex inside the cell wall. The counterstain cannot enter the cell and they will appear purple
Gram-negative bacteria
They have cell walls with a thin peptidoglycan layer. When the alcohol is added, the wall is unable to retain the crystal violet-iodine complex and it is washed away. This allows cells to be counterstained with the safranin and appear pink
How to characterize the results from each stain:
Gram-staining = two different colored stains. (1) purple staining indicates the bacteria are gram positive, they have retained the crystal-violet stain. (2) relish-pink staining indicates gram-negative bacteria, they have retained the safranin stain different shapes and arrangements (cocci/round from bacillus/rod. or bacteria that form strands or aggregate in clumps or singly) Capsule staining = bacterial cells will be stained purple, the background of the slide is darkly stained. against the dark background, the capsules of the bacteria, if present, will appear as a clear halo around the cells Endospore staining = vegetative cells will be stained red by the safranin if endospores are present, these will retain the malachite green stain and appear blueish-green
The most important step of the Gram stain is the ______
Decolorization It is possible to over-decolorize, meaning if too much alcohol is applied the primary stain will eventually be removed from the gram-positive cells and they will then appear gram-negative. The reverse is also true: if the smear is under-decolorized, gram-negative bacteria will lose the primary stain; hence they will not pick up the colors counterstain and will remain purple
Serial dilution
Adding 1 ml to a 9 ml broth Tube 1: tube dilution = 1/10 Tube 2: tube dilution = 1/10 —> final dilution = 1/10^2 (bc (1/10)*(1/10)) Tube 3: tube dilution = 1/10 —> final dilution = 1/10^3 (bc (1/10)*(1/100)) Tube 4: tube dilution = 1/10 —> final dilution = 1/10^4 Tube 5: use 0.2 ml of culture and plating on agar plate See example for calculation in pics
In the two differing pics
Both are spread plates. Did a serial dilution and used spread plate technique to spread bacteria sequentially for each dilution. The circle is done incorrectly-not spread bacteria evenly
Colony observations (check pic)
Form: what the colony looks like Elevation: Margin: ~filamentous = fungi
Differentiate between positive staining and negative staining (for capsule staining)
POSITIVE STAIN: - appearance of cell is colored by dye - background appearance is not stained - examples include methylene blue and crystal violet NEGATIVE STAIN / background staining: - appearance of cell is clear/colorless - background appearance is black/dark - examples include india ink and nigrosin - heat fixatio is not used (capsule stain)
How to qualitatively and quantitatively evaluate a sample containing a known bacterium and microbial communities found in a winegradsky column via serial dilution, spread plating, and streak plating
MEDIA PREPARATION: (1) PPE and sterilize table with ethanol (2) gather two Erlenmeyer flasks. Label one broth and the other agar (3) prepare LB agar solution (agar+agar+water) and LB broth (LB media+water) (4) after autoclaving the flasks, place them in a 40-50 C water bath (5) prepare 3 aliquots of the broth solution and label (6) gather and label 10 Petri dishes (7) pipet prepared agar onto the dishes and allow to solidify DILUENT PREPARATION: (1) wipe bench top with ethanol (2) label 10 test tubes and place in a rack (3) pipet 9 ml of saline into each tube and cover each loosely, and transfer to an autoclavable test tube rack (4) after cycle is complete, allow to cool and then store at room temp until reaching 22 C CULTIVATION OF THE TARGET ORGANISM: (1) inoculate 100 ml of solution 0 with the single colony from a previously streaked plate (2) cover tube and incubate overnight at 37C (3) to evaluate the regions of a winegradsky column, add 1 g of material from the aerobic zone to test tube 1 and mix (vortex) (4) repeat this process with 1 g of material from the anaerobic zone SERIAL DILUTION AND SPREAD PLATING: (1) remove tube containing solution 0 inoculated by the target organism from incubator and shake (2) pipet 1 ml of the solution into test tube and vortex (3) remove 1 ml of solution from the tube in step 2, and transfer it to another tube, vortex to mix (4) repeat this process through to tube 10 (5) to evaluate the aerobic and anaerobic zones of the winegradsky column, remove 1 ml of solution from each of the previously prepared tubes (in cultivation of target organism section) and transfer to the appropriate second tube (6) continue the serial dilutions through to the 10th tube TO SPREAD PLATE.... (7) pipet 100 microliters if diluted sample from each test tube 3 onto the corresponding Petri plate (8) use a sterile spreading rod to gently distribute the sample onto the Petri dish and replace the plate lid and reflame the rod (9) repeat this process for test tubes 6 and 9 dilution (10) incubate the plates containing aerobic organisms in 37C for 24 hrs. Incubate the plates containing anaerobic organisms in an anaerobic chamber set to 37C for 24 hrs (11) remove the T3, T6, T9 dilution plates from incubator and anaerobic chamber STREAKING: (1) working with one plate at a time, glide a sterile inoculating loop across the top of the media in a zigzag pattern (2) replace the Petri dish lid (3) rotate the plate by 1/3 and sterilize the loop (4) reduce the frequency of the zigzag pattern (5) repeat steps once more (6) repeat this method for the remaining plates (7) properly incubate the plates DATA ANALYSIS AND RESULTS: (1) cultures were harvested from anaerobic and aerobic zones of a winegradsky column (2) the cultures were serially diluted prior to streaking and spreading on LB agar plates (3) **streaking revealed a mixed population from each of the evaluated zones (4) ** spread plates revealed similar results ~~** a plate streaked from a mixed population will result in bacterial colonies of different shapes, sizes, textures and colors ~~** streaked and spread plates containing a known organism demonstrate a homologous population
Define capsule stain
Targets bacteria that produce a thick slimy layer surrounding their cell walls looks like a halo around the bacteria capsules typically consists of multiple polysaccharides, which can be important for pathogenicity, biofilm formation, virulence, etc. (without, may not be able to cause infection) different diameter of capsules can indicate if the bacteria is going to be more/less infectious, form better biofilms... most capsules are water soluble. Capsules do not like heat (will dry out) The protocol for performing a capsule stain is as follows: Stain the background of a clean slide with the dark primary stain (ex. india ink or nigrosin) Mix bacterial sample into primary stain Create a thin smear across the sample slide using the edge of another clean slide Air dry sample to fix to slide, but do not use heat Apply secondary stain to visualize bacterial cells Rinse briefly with water to ready slide for microscope viewing To examine if a cell has an external capsule, a capsule stain is used: capsules are nonionic and have the tendency to repel stain (so simple staining does not work) a negative staining technique is used - first stains the background with an acidic colorant such as congo red, before the bacterial cells are stained with crystal violet. this leaves any capsule present as a clear halo around the cells How to capsule staining: (1) PPE and ensure glass slides are clean (2) prepare the solutions to make 1% crystal violet solution (mix 0.25 g crystal violet powder with 25 mL of distilled water and vortex until dissolved), and 1% congo red solution (mix 0.25 g of congo red powder with 25 mL of distilled water and vortex until dissolved) (3) pipette 10 micrometers of the congo red solution onto the slide (4) using a sterile pipette tip, select a single bacterial colony from the LB agar plate and smear the colony into the dye to produce a thin, even layer (5) completely air dry the slide (5-7 min) (6) flood the smear with enough 1% crystal violet to cover the smear and let sit for 1 min (7) hold slide at angle and gently squirt a stream of water onto the top of the slide, taking care not to squirt the bacteria directly (8) continue holding at a 45 degree angle until completely air-dried (9) add a drop of immersion oil directly to slide and examine the slide in a light microscope with a 100x oil objective lens
Define the vista technology - Petri dish streaking system
Unload/load —> Open —> orient —> transfer —> streak —> stack —> unload/load (1) load pre-inoculated Petri dishes (2) power on (3) cycle on ~ drops next Petri dish into position where the dish is opened and rotated to find the inoculation point. Where is is then transferred to the streaking station (4) at the streaking station four isoloops are used for sequential 4 quadrant streaking, which improves consistency and isolation
Define the purpose of staining
Used to better view organisms under the microscope They "color" the specimens Most micro organisms are transparent and by staining we can give a color contrast between the cells in the background to better view/study the cells
define bacteria
microscopic organisms that have many distinguishing characteristics - shape, arrangement of cells, whether or not they produce capsules, and whether or not they form spores. these characteristics can be identified by staining
Describe the two main categories of culture media
(1) *Liquid broth media* - three types: (1)chemically defined: exact nutrients for species. (2)complex: cheap and easy (tryptic soy broth, yeast extract). (3)enriched: fastidious organisms, used for bacterium hard to grow like mycobacterium tuberculosis or gonneheria (blood plate, tryptic soy broth+more nutrients, complex+more) (2) *solid media* - three types: (1)agar deeps (flat inside a tube). (2)agar slants (slanted inside a tube). (3)agar plates
Define the different kinds of stains
(1) DIRECT STAIN: stains the specimen, but not the background. The light passes through the background and the colored cells are easily visible --How to make a direct (simple) stain of a bacteria (using basic dye - crystal violet) (1) using a fixed, air-dried smear of bacteria, set slide on staining rack and take crystal violet (a basic dye) and cover the smear with a few drops (2) let sit for one minute (3) wash off the excess dye with ionized water (4) blot the slide dry with blotting/bibulous paper now it is ready to be viewed under the microscope (2) NEGATIVE STAIN: stains the background, but not the specimen. The background will be colored, and the light will pass through the cells --How to make a indirect (background/negative) stain of a bacteria (acidic dye - nigrosin) (1) set slide on staining rack and add 1 drop of nigrosin near the top of theslide (2) using a sterilized inoculating loop, mix bacteria into the nigrosin (3) angle the slide, and using the edge of another microscope slide, lift and spread the dye by tapping and dragging with the second slide (4) place the initial slide back down - look for alternating dark and light (5) let air dry ready to be viewed under microscope (3) SIMPLE STAIN: Stains a bacterial smear with a single dye. The cells are colored against an unstained background. This type of stain is primarily used to determine the morphology, size, and arrangement of bacterial cells (4) DIFFERENTIAL STAIN: - A stain that uses at least three chemical reagents on a smear. - ex. differentiates between gram positive and gram negative cells. - This will generally give you more information on a bacterium compared to a simple stain. Reagents used in a differential stain are: - primary stain: initially gives color to all cells - Decolorizer: used to give a color contrast, based on the chemical make up of the cell. Will usually decolorize one type of cell while the other cell type maintains the primary stain color - Counterstain: gives a contrasting color from the primary stain to cells that were decolorized **If the cells were not decolorized, they cannot assume the color of the counterstain and will remain the color of the primary stain. *If the cells were decolorized they will pick up the counterstain. ... This type of staining is based on the chemical composition of cellular components and can be used to differentiate cells based on the color they stain
The most famous differential stain is the __(1)____. another form of differential staining is ___(2)___ Two stains that are Specialized stains are ___(3)___, ___(4)___, and ___(5)___
(1) GRAM STAIN - developed by Hans Christian gram and is used to differentiate bacteria based on their cell wall composition ; the amount of peptidoglycan in the cell (gram positive have a thicker peptidoglycan layer&missing outer membrane which allows positive to retain the crystal violet. gram negative have a thin peptidoglycan layer) - differentiates most bacterial species into gram-positive and gram-negative bacteria - four reagents used in this stain: ...(1) a primary stain (crystal violet) ...--rinse with water... ...(2) a mordant (grams iodine) ...(3) decolorizer (alcohol. ethanol) ...(4) counterstain (safranin) (2) ACID-FAST STAIN (3) endospore stain (4) capsule stain (5) flagellar stain
How to streak for single colonies
(1) first label the base of the agar plate (name, date, experiment/sample name) (2) perform four sets of streaks (3) adjust the Bunsen burner to a blue flame (4) flame a wire inoculation loop until at least half of the length is red-hot (5) allow the loop to cool holding it near the flame but not in it ~To ensure your loop is cool, touch it to a blank area of the agar. If it is too hot it was sizzle and melt the agar (6) touch the sterile loop to the center of a well isolated single colony (7) make your primary inoculation in one quadrant of the plate (8) re-flame and cool your loop (9) rotate your plate a quarter turn (10) using the sterilized loop make a second set of four streaks that overlap a small area of your primary inoculum (11) re-flame and cool your loop (12) rotate your plate a quarter turn (13) make a third set of streaks in the same manner as the second, just overlapping the previous set (14) re-flame and cool your loop (15) Rotate your plate a quarter turn again (16) For the fourth set start in the end of the third set and zigzag across the remaining area of the plate. ~* do not cross into our overlap any of the other inoculated areas (17) Re-flame your loop and return it to the rack. Then turn off the Bunsen burner *A successful streak plate should yield isolated colonies in one of the four quadrants*
It is best to calculate CFUs/mL using the average colony count of 3 plates spread with the same sample and dilution factor
(Average # of colonies X dilution factor) / amount aliquoted Lastly... Isolated colonies chosen from each plate can be used in further enrichment assays to determine species identity
Pour plating vs spread plating
*SIMILAR IN PURPOSE: looks at how many colonies are in a culture - BACTERIAL QUANTIFICATION — both have the colonies fairly spread out ~~ sometimes spread plate is more efficient, and helps reduce possibility of killing/shocking the growth of the bacteria (bc of heat factor in pour plate method - 50-60 may be too high), used in making clones and isolated colonies~more common ~~ pour plate is used in food industry to look at different bacterial contamination in food POUR PLATE METHOD: (1) Inoculate empty plate with a certain amount of the bacterial culture (2) add melted nutrient agar (3) swirl to mix (4) *colonies grow in and on solidified medium* SPREAD PLATE METHOD: (1) Inoculate plate *already containing solid medium* (2) spread inoculum over surface evenly (3) *colonies grow only on the surface of the medium*
Gram stain should be prepared with fresh cultures that are between ______ - ____ hours
24 - 48 hrs As the cultures get older, gram-positive bacteria become unable to hold onto the primary stain and will appear gram-negative
Define acid fast stain
: used to differentiate bacteria that have mycolic acid; vs bacteria that do not Targets bacteria of the genus mycobacterium (have high quantities of mycolic acid). These bacteria stain poorly with the Gram stain, because of the presence of a waxy layer in their cell wall / doesnt permeate through the thick layer of mycolic acid Mycobacterium is positive acid fast bacteria, mycobacteria, have a high content of mycolic acids in their cell walls. they will appear RED nonacid fast bacteria will stain blue/green with the counterstain methylene blue or Kinyuon stain Correct Order of Steps to Perform an Acid-Fast Stain: ~starts with a smear Stain with carbolfuchsin (dyes mycobacterium) ~using steam Rinse off excess primary stain Add acid alcohol reagent (hydrochloric acid+ethanol) Rinse with water to stop the decolorization process Counterstain with methylene blue Rinse off excess counterstain Blot dry Perform microscopy TWO TYPES (based on species of mycobacterium): - Ziel-Neelson method (heat, steam) - kinyoun method (no heat) How to acid-fast stain of sputum sample (after heat fixing): (1) add carbolfuchsin to the heat-fixed slide to cover the smear and leave on for 3 min (2) rinse the slide and shake off excess (3) destain with acid-alcohol, drop-by-drop until the pink or purple stops running (4) immediately rinse and shake off excess water (5) cover the smear with methylene blue for one minute (6) rinse methylene blue and shake off excess water (7) blot gently with Kimwipe ready to be viewed under a microscope, using oil immersion to see the small acid-fast bacilli which will appear reddish and the non-acid-fast bacilli will be blue, and all background material will be blue also
Define serial dilution
A process through which the concentration of the organism (ie bacteria) is systematically reduced through successive resuspension in fixed volumes of liquid diluent **the key to enumeration of bacteria. Since mixed sampled from a winegradsky column contain an unknown, often large number of bacteria Usually the volume of the diluent is a multiple of 10 to facilitate logarithmic reduction of the sample organism Ex. 1 g of sediment is first removed from the winegradsky zone of interest and added to 10 ml of an appropriate liquid medium. Then 1 ml of this first dilution is added to another tube containing 9 mL of medium. The process can be repeated until several different concentrations of bacteria have been prepared.
Define a Winogradsky column
A sample that contains multiple species/strains of bacteria
Preparation of a smear from a broth or agar culture differs
AGAR CULTURE: On solid media the bacteria will present as colonies on the surface of the agar. A drop of water is applied to the center of the slide and the bacteria from the plate are mixed with the water BROTH CULTURE: Since the bacteria are already in suspension, it is not necessary to add a water drop to the slide. The broth culture is simply applied directly to the dry slide
Define the three types of plating techniques
STREAK PLATING -three types: (1)quadrant streaks(4x). (2)zigzag streaks(1x.. doesn't help isolate much). (3)T streak(3x) - first a smear of bacteria, but each time less and less, producing one quadrant with single, distinct colonies - **streaking for isolation** - **ensures the culture is not contaminated** - it is important to sterilize in between streaks to reduce the amount of bacteria with each streak ~~ on fourth streak, do normal streaks but then spread down the center POUR PLATING && SPREAD PLATING - looks at how many colonies are in a culture - BACTERIAL QUANTIFICATION — both have the colonies fairly spread out
Define streak plating vs spread plating
STREAK PLATING: enables the isolation of bacteria within a sample ~accomplishes by introducing a diluted sample to one section of the solid medium supplemented with nutrients, which is divided into thirds. This inoculum is then spread over each third of the plate in a zigzag pattern. As different sections of the plate are streaked, crossing from the previous sample only once, the sample is spread more thinly. Meaning you only need to streak from one dilution to *achieve individual colonies* ~~after incubation, the streaked plates allow for observations of colony morphology. Information that can help differentiate between different bacterial species.***** SPREAD PLATING: Enables the enumeration of bacteria within a sample ~ an aliquot if a single sample is spread evenly over the entire surface of solid medium. Typically bc we don't know the bacterial numbers in the mixed sample, a spread plate is made for each of the dilutions (or a representative sample of them). ~~after incubation enumeration can be performed using the spread plates. **any plates with colony counts fewer than 30 should be discarded since small counts are subject to greater error. **any counts over 300 should be discarded because of colony crowding and overlapping can lead to underestimation of colony count ~~**to calculate the colony forming units (CFUs) per ml of suspension—> if the colony counts of each of these remaining dishes is recorded and multiplied by the dilution factor and then divided by the volume plates
Define a Gram stain
Separates bacteria into two main groups (gram-positive and gram-negative) based on their cell wall structure ex. E coli (gram negative), staphylococcus aureus & bacillus (gram positive) How to do gram staining: (1) PPE (2) clean a fresh microscope slide with a laboratory wipe (3) Pipette 10 micrometers of 1x phosphate buffered saline onto the first slide (4) use a sterile pipette tip to select a single bacterial colony from an LB agar plate (5) smear the bacterial colony into the liquid to produce a thin, even layer set the slide on the bench top and allow to fully dry (6) light a bunsen burner to heat-fix the bacteria using tongs, with the bacteria sire up. careful not to hold the slide in the flame too long (which could distort the cells) (7) hold the slide level and apply several drops of gram's crystal violet to completely cover the bacterial smear. allow to sit for 45 seconds (8) hold slide at an angle and gently squirt a stream of water onto the top of the slide, ensuring not to squirt the bacterial smear directly (9) holding the slide level, apply gram's iodine solution, allow to stand for 45 sec (10) carefully rinse the slide as done previously (11) while holding the slide at an angle, add gram's decolorizer to the slide, allowing it to run down over the stained bacteria until the run off is clear (5 sec) (12) immediately rinse with water as shown previously (13) holding the slide, level, apply gram's safranin counter-stain to completely cover the bacteria, let sit for 45 sec (14) gently rinse as shown previously (15) blot dry with paper towels (16) add a drop of immersion oil directly to slide and examine the slide in a light microscope with a 100x oil objective lens
How to prepare and sterilize culture media using system media preparation systems and how to dispense the sterilized media into Petri dishes using systec media fill automated plate pourer
Systec media prep is available with sizes ranging from 10 L - 120 L Culture media is prepared and sterilized in a removable, stainless steel bucket. ~a magnetic stirrer ensures homogeneous mixing of the media A temperature sensor controls the temp of the media throughout the sterilization cycle. ~to start a sterilization cycle, push door shut and select a sterilization program After sterilization, cool to a "user-defined" holding temperature; keeping the media liquid Once the holding temp is reached, closing caps for the adding and dispensing ports can be opened. ~heat sensitive additives can be introduced to the media To dispense the media, connect a silicon tube and select the proper program Can fill the full plate, half plate (biplate), or three sections (triplate) Can label the Petri dishes during the filling process **enables easy, practically hands, free Petri dish preparation
. What is does a clear halo around a cell indicate when bacteria are stained with Congo red followed by Crystal Violet?
The presence of a capsule
When staining a known acid fast organism, in the presence of acid alcohol decolorizer, carbolfuchsin dye should _____ in the sampled bacteria.
be retained phenol in carbon fuchshin has affinity towards mycolic acid fast organism, thus they get stained with primary stain but whereas acid fast organisms resists decolorization by acid-alcohol and remains colored (red)
Define flagellar stain
can be used to see flagella on E coli flagella are thin and delicate so they cannot be gram stained
To examine cell shape and arrangement: use gram-staining
crystal violet is added to a bacteria that has been heat fixed onto a slide a decolorized is then applied and any bacteria with a thick peptidoglycan layer will stain purple (gram-positive) gram negative have a thinner peptidoglycan layer and will lose color with the decolorizer they will stain pink when a counterstain (safranin) is added, which binds to a lipopolysaccharide layer on their outside once stained, the cells can be characterized by their morphology, size, and arrangement (chains, clusters) which aids in classification and identification
Define endospore stain
endospore: dormant, tough, non-reproductive, ensures survival of bacteria through periods of environmental stress. not all bacterial cells produce endospores distinguishes between active metabolic cells and dormant strucutres Targets bacteria of the genus bacillus (ex. anthrax) and clostridium (ex. botulism and tetanus). These bacteria produce heat resistant structures that are tough to stain otherwise bacillus serious & bacillus anthraces gram stain would not indicate how many of the bacteria have turned into its spore state To determine if the bacterial cell forms spores, endospore staining is used: difficult to stain because they are impermeable to many dyes the schaeffer-fulton method uses malachite green stain which is applied to bacteria fixed to a slide the slide si then washed with waters before being counterstained with safranin vegetative cells will appear pinkish-red, while any endospores will appear green How to endospore staining - Schaeffer-fulton method (1) PPE (2) prepare a 0.5% malachite green solution by mixing 0.125 g of malachite green powder with 25 mL of distilled water and then vortex until dissolved (3) pipetter 10 micrometers of 1x PBS onto the slide (4) using a sterile pipette tip, select a single bacterial colony (5) smear the bacteria into the liquid to produce a thin, even layer (6) allow to air dry fully (7) once dried, light a bunsen burner to heat fix the bacteria through the blue burner flame using tongs, with the bacteria side up. careful not to hold the slide in the flame too long (which could distort the cells) (8) once the slide has cooled, place a piece of precut lens paper over the heat-fixed smear (used to keep the dye from evaporating too quickly and to allow for more contact time between the dye and spores) (9) turn on hotplate to highest setting and bring beaker of water to a boil (10) saturate the lens paper with the malachite green solution (11) using tongs, place the slide on top of the beaker to steam for 5 minutes.. keeping the lens moist by adding more dye one drop at a time as needed (12) using tongs, pick up slide from beaker and remove and discard the lens paper (13) allow to cool for 2 minutes (14) hold the slide at an angle and gently squirt a stream of water onto the top of the slide (15) hold the slide level and apply safranin to completely cover the slide and let stand for 1 minute (16) hold the slide at an angle and gently squirt a stream of water onto the top of the slide (17) allow slide to air dry (18) add a drop of immersion oil directly to slide and examine the slide in a light microscope with a 100x oil objective lens
gram positive ; gram negative ; acid fast
gram positive: cells with thick layer of peptidoglycan and teichoic acids gram negative: cells with both inner and outer membrane, as well as a thin layer of peptidoglycan acid fast: cells contain thick later of mycolic acid or cord factor
How to prepare a specimen to be stained -- PREPARATION OF A SMEAR
prepare a smear. A sample of bacteria is applied to a slide in allowed to dry so staining techniques can be performed. In order to "hold" your specimen in place, so you can apply the stain and wash to your slide you must "fix" or "glue" the cells the slide, this is known as fixation Heat can be used to fix our cells to the slide (*heat-fixation*) by passing the slide through a flame 2 to 3 times. A disadvantage to heat-fixing is that this kills the cells; hence you are not able to view the cells in their natural state. The process of heat-fixing denatures cellular proteins, which allows the amino acid side chains to adhere to the slide. Once you have produced a heat-fixed smear you are ready to perform your stain STEPS: Appropriately label slide Apply bacteria to the slide using aseptic technique Air drying Heat fixation Apply staining method How to prepare a smear and carry out a simple stain: before staining, prepare a material smear ~ (1) label a glass slide at one end with the culture name (2) transfer a small drop of water aseptically onto the center of the slide using a wire loop (3) aseptically, take a small quantity of a bacterial colony from a agar plate from which the culture of interest is growing (4) transfer this culture to the water drop on the slide - spread until the water and colony form an emulsion (5) set down the slide and allow the slide to air dry (6) once completely dry, heat-fix the smear by carefully passing the slide, smear facing upwards, through the bunsen flame three times - do not overheat or hold the slide on the flame the smear is now ready to be stained