Chemotherapy

Introduction:

      Antimicrobial chemotherapy uses chemicals to inhibit or kill microbes, and is based on selective toxicity.  The goal is to inhibit or kill the organisms targeted without seriously harming the host.  This is accomplished by interacting with a microbial structure or function that is not present or is significantly different than what occurs in the host.  In prokaryotic bacterial infections, this is frequently done by targeting the peptidoglycans or bacterial ribosomes which are not present in the human host.  Eukaryotic organisms such as parasites or fungi, have structures and functions more similar to humans, and therefore the number of agents effective at inhibiting or killing those organisms without significantly harming the host is relatively limited.  Viruses are not cells and do not have the structures to be targeted by antibiotics, and therefore are not treated with antibiotics.

      Antibiotics are substances which are produced as a metabolic product of one microorganisms which inhibit or kill another microorganism.  Antimicrobial chemotherapeutics chemicals are synthesized in the lab to be used therapeutically on microorganisms.  May antibiotics today are semisynthetic and modified or even completely synthesized in the lab..  The three major groups that are used to produce useful antibiotics are the actinomycetes (filamentous soil bacteria), the Bacillus genus, and the saprophytics molds penicillium & Cephalosprium.

      Cidal agents kill microorganisms such as the penicillins, cephalosporins, streptomycin, and neomycin, while other are static in action and only inhibit microbial growth.  Those that inhibit microbial growth allow for the body’s own defenses to attach the organisms, such as the tetracyclines, erythromycin, and sulfonamides.  Drugs that target a variety of both gram positive and gram negative organisms are said to be broad-spectrum (tetracycline, streptomycin, cephalosporins, ampicillin, sulfonamides), while those that only target gram positive or gram negative are narrow spectrum (penicllin G, erythromycin, clindamycin, gentamicin).  Whenever possible, it is preferred to use the more narrow spectrum antibiotic to preserve the body’s natural flora , reduce opportunistic infections, and decreases selection for resistant strains of microorganisms.

1.     Antimicrobial agents that inhibit peptidoglycan synthesis.  This results in lysis of actively dividing bacteria.

a.      penicillins

                                                        i.     Natural penicillins – active against gram positive, but are inactivated by penicillinase (Penicillin G, F, X, K, O, V)

                                                      ii.     Semisynthetic penicillins – active against gram positive but not inactivated by penicillinase (methicillin, dicloxacillin, nafcillin)

                                                     iii.     Semisynthetic braod-spectrum penicillin – both gram positive & gram negative but inactivated by penicillinase (ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, piperacillin)

                                                     iv.     Semisynthetic broad-spectrum penicillins with beta lactamase inhibitors – the beta lactamase inhibitors inhibit the penicillinase enzyme

b.     cephalosporins – active agains a variety of gram positive and negative organisms and resistant to penicillinase  (cephalothin, cephapirin, and cephalexin, cefamandole, cefaclor, cefazolin, cefuroxime, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, deftazidine, moxalactam)

c.      carbapenems – broad spectrum beta lactam to inhibit peptidoglycan synthesis (imipenem)

d.     monobacterms – broad spectrum beta lactam resistant to beta lactamase (aztreonam)

e.      carbacephem – synthetic cephalosporin

f.      glycopeptides – target gram positive bacteria (vancomysin, teichoplanin)

g.     bacitracin – targets gram positive, used topically

h.     fosfomycin

2.     Altering the cytoplasmic membrane

a.      Polymyxin B – used in severe Pseudomonas infections

b.     Amphoterocin B – systemic fungal infections

c.      Nystatin – Candida infections

d.     Imidazoles – antifungals (clotrimazole, miconazole, ketoconazole, itraconazole, fluconazole)

3.     Agents that inhibit protein synthesis

a.      Agents that block transcription (rifampins) inhibit RNA polymerase, some gram positive and negative, M. tuberculosis

b.     Agents that block translation – (streptomycin, kanamycin, tobramycin, amikacin, tetracycline, minocycline, doxycycline, erythromycin, roxithromycin, clarithromycin, azithromycin) – target gram positive and gram negative, (lincomycin & clindamycin) – usually used against gram positive, (oxazolidinones, streptogramins)

4.     agents that interfere with DNA synthesis

a.      fluroquinolones – inhibits enzymes called topoisomerases needed for bacterial nucleic acid synthesis (norfloxacin, ciprofloxacin, enoxacin, levofloxacin, trovacloxacin)

b.     sulfonamides & trimethoprim – block enzymes needed for synthesis of tetrahydrofolate to make bases thymine, guanine, uracil, & adenine

c.      metronidzaole – nicks microbial DNA strands

Methods of microbial resistance:

1.     enzymes that detoxify or inactivate the antibiotic

2.     altering the target site or reduce / block the binding site of antibiotic

3.     prevent transport of agent into the bacterium

4.     develop alternate metabolic pathway to by-pass step blocked by the agent

5.     increased production of certain enzymes

These changes occur naturally that allow a bacterium to resist the antimicrobial agent as the result of mutation and genetic recombination.  With exposure, the resistant organisms are selected for because the others die out, leaving only the resistant organisms to multiply.  These bacteria may also transfer resistance to other bacteria, both of similar and different genus & species by the use of R plasmids.  R (resistance) plasmids have genes coding for multiple antibiotic resistance, and are transferred between bacteria through the use of a sex pilus.  The normal flora may acquire the R plasmids from pathogenic bacteria.  Resistant normal flora may be transmitted person to person as well.  Antibiotic susceptibility testing can be done in the clinical laboratory to determine which antimicrobial agents will most likely be effective at targeting that particular strain of organism.

In some cases the susceptibility to a chemotherapeutic agent is predictable, but in many cases there is no reliable way of predicting which antimicrobial agent will be effective, especially with the emergence of so many antibiotic resistant strains.  This may be tested using tube dilution tests, the agar diffusion test (Bauer-Kirby test), and automated tests.  The automated tests are able to give results within a few hours, but equipment is very expensive.

Tube dilution tests are performed using a series of cultures with different concentrations of the chemotherapeutic agent.  The tubes are inoculated with the test organism and incubated 16-20 hours and examined for growth.  The MIC (minimum inhibitory concentration) is the lowest concentration of the chemotherapeutic agent that prevents growth of the test organism.  Additional tests to determine MBC (minimum bactericidal concentration) to determine the lowest concentration of a chemotherapeutic agent that inhibits growth of the cultures, are time consuming and expensive.

Procedure:

In this lab we will be using the Bauer-Kirby disc diffusion method.  The standardized antibiotic-containing disc has been correlated with the clinical response of patients given that drug.  Zones of growth inhibition are correlated with the MIC for each agent to establish categories of “resistant”, “intermediate”, and “sensitive”.

1.    Prepare a standard Mueller-Hinton agar plate with a standardized inoculum covering the entire agar surface with bacteria. Also prepare 1 plate with a throat culture collected using a sterile swab. Cover the entire plate.

2.    Place standardized antibiotic-containing discs on the plate.

3.    Incubate the plate for 18-20 hours at 35 degrees Celsius.

4.    Measure the diameter of any resulting zones of inhibition in millimeters.

5.    Determine if the bacterium is susceptible, moderately susceptible, intermediate, or resistant to each antimicrobial agent using a standardized table.  (Zone Size Interpretive Chart for Bauer-Kirby Test)

 

Prepare a table of your results:

Organism	Chemotherapeutic agent	Zone size (mm)	Sensitivity
Zone of Inhibition Example:	http://www.life.umd.edu/classroom/bsci424/Images/PathogenImages/AntibioticZonesofInhibition.jpg

 

Chemotherapy

Disinfectants & Antiseptics

Introduction:

In many different industries the elimination of microorganisms from inanimate objects or surfaces is critical to safety.  (Please review terminology covered in Lab 6).  Disinfectants and antiseptics often work slowly on certain viruses and endospores may be difficult to destroy, sterilization may not be practical.  Results are generally better when initial microbes numbers are small and the surfaces being disinfected are relatively free of interfering substances.   Disinfectants and antiseptics may damages the lipids / proteins of the cytoplasmic membrane of the organisms resulting in the cell leaking, or they may denature enzymes and proteins; affecting metabolism.

Common chemical agents:

1.     Phenol and phenol derivatives.  Phenols were the first disinfectants commonly used, but due to toxicity and odor, phenol derivatives are now generally used.  These alter membrane permeability and denature proteins.  These kill most bacteria & fungi, some viruses, but are generally ineffective against endospores.

Examples:  Othophenylphenol – Lysol, O-syl, Stpahene, Amphyl

Hexachlorophene – PhisoHex

Triclosan – antimicrobial soaps

Hexylresorcinol – thoat lozenges

Chlorhexidine – handwashing / surgical scrub

2.     Soaps & Detergents:  Soaps are mildly microbicidal and aid in the mechanical removal of microbes by emulsifying the oily film on skin and reducing the surface tension of water.  Anionic detergents mechanically remove microbes and other materials but are not very microbicidal.  Cationic detergents alter membrane permeability and denature proteins.  They are effective against many vegetative bacteria, some fungi and viruses, but endospores are generally resistant.  They are inactivated by soaps and some other organic materials.

Examples of cationic detergents:  benzalkonium chloride, zephiran, diaprene, roccal, ceepryn, phemerol

3.     Alcohols:  (ethyl, isopropyl):  denature membranes, often combined with other disinfectants, usually kill vegetative bacteria, enveloped bacteria, & fungi, do not generally kill endospores or non-enveloped viruses

4.     Acids / Alkalies:  alter membrane permeability, denature proteins / other molecules, organic acid salts such as calcium propionate, potassium sorbate, and methyl paraben are used as food preservatives, undecylenic acid (Desenex) for skin infections, lye (sodium hydroxide) in soap

5.     Heavy metals:  (Hg, Ag, Cu):  denature proteins, mercury compounds are bacteriostatic & do not skill enodspores, silver nitrate is used to prevent neonatal gonococcal ophthalmia, copper sulfate is used as a plant fungicide, and selenium sulfide is used as a fungicide

6.     Chlorine:  denatures enzymes, sodium hypochlorite is in household bleach, sodium hypochlorite & chloramines are used to sanitize glass & food equipement.

7.     Iodine & iodophores:  denature proteins, effective against vegetative bacteria, Mycobacterium tuberculosis, fungi, some viruses & endospores, (Wescodyne, loprep, loclide, betadine, isodine)

8.     Aldehydes:  denature proteins, formaldehyde & glutaraldehyde, formalin kills most microbes and is used in embalming, vaccines, and preparation of biological specimens, glutaraldehydes kill vegetative bacteria & endospores in 4 hours and are used in cold sterilization (Cidex, Sonacide, Sporocidin)

9.     Ethylene oxide:  denatures proteins, used in chemical sterilization after 4-12 hours, used for head sensitive items, very toxic and carcinogenic

Procedures:

Procedure:

Is that surface as clean as you think it is?

1.    Swab a surface you intent to clean using a sterile swab, and then inoculate half of the plate.

2.    Use the household cleaner you wish to test to clean the surface as you would normally use the product.

3.    Swab the cleaned surface using a sterile swab & then inoculate the other half of the plate.

Are you sure your hands are clean?

1.      Divide a plate of trypticase soy agar in half.  Inoculate half the plate using your finger or a swab from your uncleaned hand.

      2.  Use your favorite hand sanitizer to clean your hands as you normally would.

      3.  Repeat the procedure used in step one to incoluate the other half of the plate with your “sanitized” hand.

To kiss or not to kiss?

1.      Divide a plate of trypticase soy agar in half.  Innoculate the entire plate using a sterile swab rubbed on one of your teeth.

      2.  On 1 half of the plate, use a sterile swab to apply your favorite tooth paste or mouthwash to 1 half of the plate.  The other side will be a control.

      3.  You may wish to run a separate control swabbing a sample of your tooth paste or mouthwash since these items are frequently contaminated by your toothbrush or taking mouthwash directly from the mouth of the bottle.

Expected Results:  

Sorry, I enjoy seeing the look on your face way too much to give this one away!

Microbial Control

Introduction:

Growth of microbial agents are controlled through the use of physical agents & chemical agents.

Basic Terminology:

      Sterilization:  process of destroying all living organisms & viruses, including endospores

      Disinfection:  elimination of microbes from inanimate objects / surfaces

      Decontamination:  treating objects or inanimate surfaces to make them safe to handle

      Disinfectant:  used to disinfect inanimate objects, generally too toxic to use on human tissues

      Antiseptic:  agent that kills / inhibits growth of microbes, safe to use on human tissue

      Sanitizer:  agent that reduces but may not eliminate microbes numbers to a safe level

      Antibiotic:  metabolic product of one organism that inhibits / kills another microbe

      Chemotherapeutic antimicrobial agent:  synthetic chemical to inhibit / kill other microbes

      Cidal:  action will kill microorganisms / viruses

      Static:  agent will inhibit growth of microorganism

Use of temperature:

      Microorganisms have an optimum, minimum, and maximum temperature for growth.  Temperatures below the minimum usually have a static action, while those above the maximum tend to have a cidal action.  High temperatures can generally kill vegetative organisms from 50 to 70 degrees Celsius with moist heat because it is able to penetrate microbial cells.  Bacterial endospores are very resistant to heat and may require extended exposure.  Heat may denature proteins & melt lipids.  Dry heat kills microbes through protein oxidation.

1.     Autoclaving:  uses steam under pressure, so the temperature is raised to 121 degrees Celsius under 15 psi.  This is sufficient to kill endospores.

2.     Boiling water:  100 degrees Celsius, generally kills vegetative cells after 10 minutes, but certain viruses such as hepatitis may survive up to 30 minutes, and endospores may even survive hours of boiling.

3.     Hot air sterilization:  ovens use high dry temperatures such at 171 degrees Celsius for 1 hour, 160 degrees Celsius for 2 hours, or 121 degrees Celsius for 16 or more hours, and generally used for glassware & instruments.

4.     Incineration:  used to destroy disposable materials by burning.

5.     Pasteurization:  mild heating to kill particular organisms or pathogens (used for milk), but does not kill all organisms, milk heated to 71.6 degrees Celsius for at least 15 seconds or 62.9 degrees Celsius for 30 minutes

6.     Low temperature:  inhibits microbial growth by slowing metabolism, 5 degrees Celsius slows growth for food for a few days, freezing at -10 degrees Celsius stops microbial growth but generally does not kill organisms.

Other methods to control microbial growth:

1.     Dessication:  this has a static effect.  Microbial enzymes are inhibited by the lack of water.  This is the method used in freeze-dried foods.

2.     Osmotic pressure:  this is a method using the concentration of water and dissolved materials to alter the natural environment of the organisms and inhibit growth.  Water will move from the greater water and lower solute concentration to the area with lesser water and greater solute concentration.  An environment where the solute is higher in the cell than outside the cell, the environment in the cell is hypotonic and water will flow into the cells.  Rigid cell walls of bacteria and fungi prevent bursting of the cell.  If the solute is higher outside the cell than inside the cell, the environment inside the cell is hypertonic, and water will flow outside the cell.  When the solute is the same on both the inside and outside of the cell, the environment is isotonic.  When the inside of the cell is hypertonic, water will flow out and the cell becomes dehydrated, inhibiting growth.  This is the action of canning jams or preserves with a high sugar concentration.  Mold tends to be more tolerant of hypertonic conditions and thus require sealing to exclude oxygen (molds are aerobic).

3.     UV radiation:  This includes wavelengths from 100 to 400nm.  The cidal activity depends on the length of exposure and the wavelength used.  The most cidal wavelengths are 260-270nm where it will be absorbed by nucleic acids.  Mutations caused by UV radiation can leady to faulty protein synthesis, and if they are sufficient it may block metabolisms and kill the organism.  UV light has poor penetrating power and is only effective for microbes on the surface.  It may also damage the eyes, cause burns, and cause mutations in human cells.

4.     Ionizing radiation:  this includes x-rays and gamma rays, and have greater penetrating power.  This type of radiation can disrupt DNA and proteins, and is used to sterilize pharmaceuticals, disposable supplies, and in certain foods.

5.     Filtration:  these may be used in cases where the filters may have small enough pores to allow organisms-free fluid to pass and prevent the passage of microbes.  Filters have pores ranging from 25nm to 0.45micrometers.  This technique is not effective for viruses which are able to fit through the pores.

 

 

 

 

Quantifying Bacteria

Introduction:

     It is often necessary  to determine the number of bacteria in a sample or compare bacterial growth.  Quantifying organisms has particular importance in the food and water industry.  A variety of methods are available to determine approximately how many organisms are present in a suspension.  The method used will depend on whether total counts or counts of only viable organisms needs to be measured.

The Plate Count (Viable Count):

     Usually the number of bacteria in a given sample is too great to be counted directly.  In these cases, the sample is serially diluted, and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies.  The number of colonies can be used to measure the number of viable cells in that known dilution.  Because some organisms form multiple cell arrangements, the colony may consist of groups of bacteria rather than a single organisms.  We generally refer to these as colony forming units (CFUs) in that known dilution.

     Samples are generally diluted by factors of 10 and plated on agar.  The number of colonies on a dilution plate, after incubation, will be between 30 and 300.  This range is chosen because it is statistically significant.  Small dilution errors will have a drastic effect on a plate with less than 30 organisms, while with more than 300 organisms there would be poor isolation and colonies growing together.  Usually, you will determine the number of CFUs per milliliter of sample.  This will be done by finding the number of colonies on a suitable plate and multiplying it by the dilution factor.

     # CFUs per ml sample = # colonies (30-300 plate) X dilution factor of plate counted

Direct Microscopic Method (Total Cell Count):

     In the direct microscopic count, a special coverslip with a ruled slide is used the count the cells in a known volume.  The number of bacteria is counted directly under the microscope, and then the number of the bacteria in a large sample is calculated by extrapolation.  This is useful to count both living and non-living organisms.

Turbidity:

     Since growth of microorganisms in a liquid medium causes it to become turbid, the amount of light absorbed by the bacterial suspension can be used to estimate the number of bacteria present in the sample.  The instrument used is a spectrophotometer, which only allows a single wavelength of light to pass through the sample.  A photocell compares the light coming through the sample with the total light entering the tube.  The percent of light transmitted in inversely proportional to the bacterial concentration., while the absorbance is directly proportional to the cell concentration. 

Direct Microscopic Count:

     In this technique, 1.0ml of the sample is pipetted into a tube containing 1.0ml of the dye methylene blue to give a ½ dilution of the sample.  A Pipette is used to fill the chamber of a Petroff-Hausser counting chamber (a special slide with a counting grid) with this ½ dilution.  A cover slip is places over the chamber and the slide is placed under the 40X objective.  The number of bacteria are counted in 5 of the large double lines squares.  For more accuracy, organisms on the upper and left lines are counted, but not on the right and lower lines.  This total number is divided by 5 to find the average number of bacteria per large square.  The number of bacteria per cc is calculated as follows:

The number of bacteria per cc =

The average number of bacteria per large square X

The dilution factor of the large square (1,250,000) X

The dilution factor of any dilutions made prior to placing the sample
in the counting chamber, such as mixing it with dye (2 in this case).  This is essentially the same as a random sampling technique that can be used to approximate the count of other organisms (including large mammals) when counting all of them is impractical.

Procedure:  Serial Dilutions for Viable Plate Count

 

 

Procedure: - Viable plate count

You will need 6 dilution tubes containing 9.0 ml of sterile saline.  Use aseptic technique to dilute 1.0 ml of a sample of bacteria.  You will be using a new sterile pipette for each 1.0ml that you dilute.

Place 1.0 ml of sample aseptically into 9.0 ml of sterile saline.  Mix the tube thoroughly by holding the tube in 1 hand and vigorously tapping the bottom with the other hand to assure even distribution of the bacteria throughout the liquid.

Use the same procedure to aseptically withdraw 1.0ml from the first dilution tube & dispense it into the second dilution tube.  Continue to do this from tube to tube until the dilution is completed.  Be sure to use a new pipette for each dilution.

Using a new pipette, aseptically transfer 0.1 ml from each of the last dilution tubes onto the surface of the corresponding labeled trypticase soy agar plate.  Since only 0.1ml of the bacterial dilution rather than the desired 1.0ml is placed on the plate, the actual dilution of the plate is 1/10 the dilution of the tube from which is came.

Use a sterile cell spreader to immediately spread the solution over the entire surface of the agar plate.

Replace the lid and resterilize the glad rod with alcohol and flame.

Repeat this process for each of the plates.

Once the plate has dried, incubate the plates upside down at 37 degrees Celsius.

Counting the colonies:

Choose a plate that appears to have between 30 and 300 colonies.

Calculate the number of CFUs per ml of original sample as follows

CFUs / ml sample = # of colonies X dilution factor of the plate

     ___________ number of colonies

     ___________dilution factor of the plate counted

     ___________ number of CFUs per ml.

Practice Examples:

Using the following diagram, how many CFUs per ml are in the original sample?

http://www.sciencebuddies.org/mentoring/project_ideas/MicroBio_img019.gif

Examples of plates after going through series of dilutions:

Top row:  plate on the far row offers most reasonable number of colonies to count

Bottom row:  middle plate has enough to count to be valid, but not too few or too many.

Website with sample calculations:

Isolation & Gram Stain Preparation

Introduction:

     Since microorganisms live as mixed cultures in nature, we need to be able to isolate them into a pure culture for study.  The most common method to do this is the streak plate method.  In this lab you will streak 2 colonies from each of the sample plates for isolation.  The streak plate method dilutes the sample mechanically by dragging the loop across the agar surface, separating the organisms.  The loop is flamed between each region to help dilute each region further.

     Bacteria can also be isolated by using specialized media.  These help the microbiologist inhibit growth of unwanted organisms or visually identify likely organisms.  Selective media contain stubstances to inhibit the growth of 1 group while permitting the growth of others.  Columbia CNA agar contains the antibiotics colistin and nalidixic acid to inhibit the growth of Gram negative organisms.  It is selective for Gram positive organisms.

     Differential media causes an observable color change, differentiating the bacteria that are able to carry out that specific biochemical reaction.  Enrichment media enhances the growth of certain organisms.  Some media are both selective and differential.  EMB (Eosin Methylene Blue) agar is selective for Gram negative organisms and is differential for certain Gram negative enteric bacteria.

     Escherichia coli:       large blue/black colon, green metallic sheen

     Enterobacter & Klebsiella:     large, mucoid, pink/purple colonies, no metallic sheen

     Salmonella, Shigella, Proteus:  large colorless colonies

     Shigella:  colorless to pink colonies

Isolation using different media:

Select 2 different colonies from each of the finger culture, hair culture, and air culture and streak for isolation.  You will prepare a streak for isolation a plate of Columbia CNA agar and EMB agar for each sample colony. For each colony you will streak ½ of a CNA plate and ½ of a EMB plate.

Label your plates and incubate at 37degrees Celsius.

Gram stain preparation:

     Bacteria are not easily visualized under a microscope without staining.  Stains allow a greater contract between the organism and its background, allow us to identify the morphology (shape, arrangement, Gram reaction), and see flagella, capsules, endospores, etc.  Dyes are stains are generally divided into basic and acidic dyes. 

     The Gram stain is the most common stain used in microbiology, and is used to differentiate between Gram positive and Gram negative bacteria.  Gram positive bacteria stain purple, and Gram negative stain pink.  The difference in Gram reaction is due to differences in cell wall structure.  The Gram positive cell wall is 60-90% peptidoglycan.  The Gram negative cell wall only has 2-3 layers of peptidoglycan surrounded by an outer membrane of phospholipids, lipopolysaccharide, lipoprotein, and proteins.  Only 10-20% of the Gram negative cell wall is peptidoglycan.

 

Heat fixation from agar medium:

1.  Place ½ a drop of distilled water on a clean slide.

2.  Use aseptic technique to transfer a small amount of culture from the agar surface and touch it to the water until it turns cloudy.

3.  Burn the remaining bacteria off the loop.

4.  Use the loop to spread the suspension over a large portion of the slide for form a thin film, and allow it to completely air dry.

5.  Pass the slide film side up through the flame of the burner 3-4 times to heat fix.  Be careful not to use too much heat that may distort the organisms.  Too much heat may cause a Gram positive organism to stain Gram negative.

Heat fixation from a broth culture:

1.  Aseptically place 2-3 loops of the culture on a clean slide.

2.  Spread the suspension over the slide and allow to air dry.

3.  Use the burner to heat fix as described above.

Gram stain procedure:

1.  Stain the bacteria with the basic dye crystal violet.’(1 minute)

2.  Cover the smear with Gram’s iodine solution.  This allows better retention of the stain by forming an insoluble crystal violet iodine complex. (1 minute)

3.  Decolorize the smear with the Gram’s decolorizer, a mixture of ethyl alcohol and acetone.  Gram positive bacteria will retain the crystal violet complex while Gram negative are decolorized. (just until purple stops flowing, and wash with water immediately)

4.  Counterstain with the basic dye safranin.  Gram positive bacteria are not affected by the counterstain because they are already purple.  Gram negative bacteria are colorless, and will become directly stained by the safranin.  (2 minutes and then wash with water).

Evaluate your skill in streaking for isolation with the sample colonies:

Results:  Note how many different colonies it appears  from each sample colony you streaked.

 

http://inst.bact.wisc.edu/inst/images/book_3/chapter_14/14-2.jpg

Example Gram Stain Results:

Gram positive rods:

http://lib.jiangnan.edu.cn/ASM/115-8.jpg

Gram positive cocci:

http://media-2.web.britannica.com/eb-media/05/58705-004-12AFD703.jpg

Gram Negative Rods:

http://jon9783.myweb.uga.edu/image/image1.jpg

Gram Negative Cocci:

http://lib.jiangnan.edu.cn/ASM/118-1.jpg

Basic Microscopy

Introduction:

Part of the process of identifying bacteria is viewing them under the microscope.  There are 3 common shapes of bacteria.  Bacteria are prokaryotic cells and reproduce by binary fission.

Coccus: 

     spherical shape, may appear oval, elongated, or flattened

     Size:  0.5 – 1.0 microns

Diplococci:  pair of cocci, examples (Neisseria sp.)

Chains:  Streptococcus species

Tetrads:  square of 4

Sarcina:  cube of 8

Clusters:  Staphylococcus

Bacillus:  rods

Streptobacillus - in chains

Coccobacillus – oval shaped

          Size:  0.5-1.0 microns wide, 1-4 microns long

Spiral:

          Vibrio:  comma shape (Vibrio cholera)

          Spirilium:  thick, rigid, spiral

          Spirochete:  thin, flexible spiral

          Size:  width typically 0.25-0.5 microns,

               Length:  commonly 5-40 microns, some are over 100 microns

     Yeasts:  unicellular fungi, eukaryotic, spherical, reproduce by budding

          Size:  3-5 microns in diameter

Measurement can be approximated using a microscope with an ocular micrometer (an eyepiece with a scale superimposed) or approximating the size in comparison to the pointer.

 

Basic Microsopy:

Refraction:  bending of a light ray as it passes from 1 medium to the another

Index of refraction:  ratio of the velocity of light in a vacuum to its velocity in a given medium

Resolving power:  inversely related to resolution, increased by using a shorter wavelength of light, increasing the refractive index, and increasing the  aperature of the objective.  A typical microscope can resolve 2 points 0.27 microns apart, and 0.2 microns for oil (the limit of the eyes).

Immersion oil increases the refractive index, increases the angle of the aperature, and decreases the scatting of light.

 

Use of the compound light microscope: 

PLEASE CARRY MICROSCOPES CAREFULLY WITH BOTH HANDS.  Always clean off oil and turn off your microscope when putting it away.   (financial aid does not cover the cost of replacing a microscope!)

1.  There are 4 objectives – a scanning lens, a low power, a high dry, and an oil immersion objective.  The lowest power has the largest aperature and the immersion is the smallest.

2.  Turn on the light.  Use the 4X objective to scan the slide and make sure it is under the objective.  Rotate the low power 10X objective into place.  In microbiology, the 4X is rarely used.  As you become comfortable with microscopy you may begin with the 10X objective.

3.  Move the condenser all the way up.  This position is used for almost all microscopy, but can increase contract and decrease resolution in some wet mounts.

4.  Use the iris diaphragm to adjust the light intensity.

5.  Adjust the distance separating the eyepieces while looking through the microscope.  You should see 1 complete circle.

6.  Focus on the specimen.  Raise the stage as far as it will go and slowly lower the stage with the coarse adjustment until the specimen is in focus.  Always focus so that you can avoid crashing the lens into the slide!

7.  Finish with the fine adjustment.

Swing the high dry lens in place after focusing with the low power 10X objective.  The objectives should be parfocal so only minor fine adjustment is necessary.You may need to increase the light by opening the iris since the aperature is smaller in higher objectives.To focus with oil, first focus with the lower power objectives.  Open the iris and place a drop of oil directly on the stained specimen.  Swing the oil lens into place, rotating in the direction avoiding passing the high dry through the oil.  Make sure the long lens does not touch any part of the mechanical stage.  Only fine adjustment should be needed to focus on the specimen.Remove the oil from the lens with a flat piece of lens paper.  Do not leave oil on the objective as it can seep in and harm the lens.

Procedures:

For bacteria slides- view under 100x with oil

For malaria- view under 10x with oil

Most parasites may be viewed under 40x

Prepare a wet mount of your hair using a clean slide, water, and a cover slip. 

What to look for in the results?