Laboratory Notebook Record Keeping Proceduresnewburyparkhighschool.net › malone ›...

Microbiology Lab Manual Page 0

Medical Technology

Microbiology Lab Manual

Isolation and Identification of Bacteriafrom Mixed Bacterial Culturesfor Medical Diagnoses


Laboratory Notebook Record Keeping Procedures

1. Use only your official microbiology notebook to record your work. All work must be recorded in the notebook and in no other document.

2. Date and sign the top of every page.

3. Sign and date at the end of each experiment.

4. Number every page. (For consistency, the entire class will number the outside corners of their lab manual.

5. Do not tear out any page(s).

6. Maintain a Table of Contents as you make entries in the notebook. The first page of every lab investigation should be listed in the Table of Contents.

7. Make all entries in black permanent ink. No pencil entries are permitted. The use of colored pens or pencils is acceptable in some cases, as approved by the supervisor.

8. Neatness is mandatory . All entries must be legible, in your neatest writing, such that a judge or a court of law can read, comprehend, and verify your data or original thought. Use a straight edge (rulers) to create tables and graphs. Skip lines, as necessary, for clarity, so as to not crowd information.

9. Do not erase, ink over, or white out errors. Draw a single line through errors so they can still be read. Place your initials and the date next to the correction.

10. State the objective or purpose of each experiment, and reference previous work or projects.

11. To protect the credibility of the notebook, there should never be any blank pages or blank spaces below an entry. Blank pages need to be crossed out and initialed and dated. Blank areas under images, tables or graphs should be labeled “NWUI – or Nothing Written Under Insert” then initialed and dated.

12. Blank or unused pages can be labeled, “ILB – or Intentionally Left Blank” then initialed and dated.

13. Use “from…” or “go to…” statements to tie together sections of a lab report of continuous work. When procedures, data, or conclusions, etc., are continued from previous pages, each page must have a “from page___” listed. When continuing to another page, there should be a “go to___” statement directing the reader to the next page of that work.


14. Record all procedures, materials, and quantities used, plus reactions or operating conditions, in sufficient detail and clarity so that someone of equal skill could understand or repeat the procedure if necessary.

15. Avoid abbreviations and codes when possible. Only the standard abbreviations from metric measurements may be used universally. Any coding or special labeling on samples or in procedural notes should be fully recorded and explained in the notebook.

16. List all persons from whom samples or data were obtained, shared or transferred.

17. Attach as much original data as is practical in the notebook. Where it is not practical to attach original data, attach examples and make clear reference to where the original data are stored.

18. For important entries, such as key conclusions or new ideas, have a coworker sign and date the entry. Be sure the coworker is not a co-inventor, but someone who is capable of understanding the meaning of the notebook entry.

19. Write/print clearly so there is no ambiguity about the information recorded. Skip lines between data tables, graphs and important conclusions to make it easier to find and read recorded information.

20. All data, especially tables, pictures, and graphs, must be properly titled, labeled as necessary, and annotated. Indicate scale and/or magnification, as necessary, if drawing or taking pictures of organisms from a microscope. Annotations must explain what the data is about.

21. Follow proper scientific rules for drawing. (refer to your physiology notebook).

22. All data must be witnessed, dated, and signed by a coworker that is not a co-inventor.


Pre-Lab Requirements

It is an expectation that you come prepared for the labs during the microbiology series. Labs must be read and pre-labs completed before you are granted permission to continue.

1. Pre-labs are to be completed prior to each lab.

2. Regular lab entries will be completed on a new page following the pre-lab.

3. Title the pre-lab with descriptor: a. Ex. Pre-Lab 1 – Aseptic Technique and Inoculation of Medium

4. Restate in your own words or copy the purpose into the pre-lab section.

5. Read the “Background” section of your lab manual, and highlight directly in the lab manual. Then OUTLINE the background information for your pre-lab. Make sure the outline is of “appropriate” length.

6. Read and highlight the procedure directly in your notebook. (Mark the text). Then proceed by drawing a flowchart of the procedures. The flowchart should be neat, clear, and concise, with annotated notes of the steps, as shown to the image on the right. If you are not familiar with a piece of equipment or of a technique, you may need to look it up on the internet to better understand the techniques so that you can do a proper flowchart. Make the flowchart images large and clear enough, such that a fellow coworker can replicate the procedures simply by using your flowchart. Chicken scratch will not be accepted.

7. When applicable, create a “rough draft” of the kind of data table you will need to organize your results. You will share this data table with your group, and the data table that your group finally decides on may be different. You may not always need to create a data table for each lab.


Lab 1: Aseptic Technique and Inoculation of Mediums

Purpose: To practice the safe, aseptic transfer of bacterial cultures between different mediums.

Background:In nature, microorganisms exist as mixed populations of many different types. However, our knowledge of microbiology has increased through the study of isolated species, grown in environments free from contamination by other living organisms such as fungi.

It is necessary for you to gain the skills to successfully transfer a pure culture of bacteria from a stock culture source to a new, sterile medium and to do so without inoculating it with any contaminating bacteria. This skill is known as aseptic technique.

In the inoculation procedure, a metal inoculation loop is used. You will be initially transferring from a stock, broth solution into a new sterile broth tube. After this transfer, you will be transferring from the stock broth to a solid, agar medium in order to grow isolated colonies. In each of these techniques, the sterility of the inoculation loop as well as even the air in and around the culture tube is crucial. Flame will be used to sterilize the inoculation loop by heating it until it is red hot, then allowing it to cool. Laying down, waving, or blowing on a cooling loop will contaminate it. The surface over which you are working must be sterilized before and after you do this lab and never should an open container of bacteria be laid down without proper closure nor the loop set down again before it is sterilized. Leaving a culture tube or agar plate open to the environment exposes it to bacterial spores which only need to land on its surface to colonize and contaminate your cultures. Be mindful of what you are doing!

Safety/Disposal:In order to maintain a sterile environment, and to maintain a level of personal safety, do not eat or drink during labs. All lab stations will be cleaned with a 10% bleach solution, and you will need to wash your hands, using the guidelines from the World Health Organization. Cleaning of lab stations and washing of hands will be completed before and after each lab. Long hair must be tied back. Hair is one of the top contaminates in a microbiology lab.

Be careful when working with the inoculating loops as they can be very hot and will cause burns. Never point the burners towards people or yourself.

Dispose of materials as instructed. Bacteria are never disposed of in the trash, unless it has been heat-treated and killed through an autoclave process (or killed using a bleach solution). Wipe down the lab table with 10% bleach solution and wash hands thoroughly before leaving the lab.


Materials for each lab group:

Lab stoveSharpieEight nutrient broth tubesInoculating loopTest tube rack

Stock Solutions:1. Bacillus sp. 2. Staphylococcus epidermidis3. Enterococcus faecalis 4. Pseudomonas aeruginosa5. Escherichia coli6. Serratia marcescens

Procedure:

1. Obtain 7 tubes of nutrient broth.

2. Your group will inoculate each of the tubes with a separate stock culture or as per assigned by the teacher. The last tube will be your control tube. Label your tubes with the name of the bacterial culture, date, initials, and station number. (For example: Bacillus sp. 1/19/13 MA-1) Label the control tube as Control. Include the date, your initials, and station number on the control tube as well.

3. Start with the control tube. Practice aseptic technique by first flaming the loop. Remove the cap of the tube using the procedure demonstrated (Figure 1) and flame the mouth of the tube. Insert the loop as to “inoculate” the broth. Re-flame the mouth of the tube and recap it then re-flame your loop. With good technique, this tube should not have any growth. Each member of your group will repeat this procedure with the control tube.


4. The other tube should be inoculated with the bacteria. Flame the loop, letting it cool. Place both the stock culture and sterile tube in your left hand. Uncap both, and keep the caps in your right hand. Dip your cooled loop into the stock culture and carefully pull out a loop of the culture, carefully putting it into the sterile tube. Re-flame the tube openings. Replace the caps and re-flame your loop. Each person in your group will repeat this procedure.

5. Repeat the above procedure (step 4) with the remaining five stock cultures.

6. Incubate all seven of your tubes in the incubator at 37 degrees Celsius until next class, putting the tubes in the class test tube rack.


Lab 2: Aseptic Streaking of Agar Plate for Isolation of Colonies

Purpose:To isolate bacteria from each other on an agar plate into individual colonies

Background: In order to separate one type of bacteria from another and to view individual colonies, it is necessary to spread a broth culture very thinly over the surface of agar plates so that they can be separated enough to be distinguished individually. This is called the streak plate method. The objective of this procedure is to separate bacteria from each other so that they can be viewed as individual colonies. Each colony begins as one bacterium. So the separation of a small amount of the culture is crucial. These cells will start packed as a drop from the inoculation loop. As the streaking occurs, the cells are spread out farther from each other, allowing individual colonies to grow.

You will need to make very careful observations of the resulting plates you inoculate from each of the stock cultures, as you will be separating mixed cultures using two or three of these stock cultures and attempting to purify the mixture in a few weeks (ONLY IF TIME PERMITS)

Materials for each lab group:

Lab stoveSharpieSix nutrient agar platesInoculating loop

Stock Solutions:1. Bacillus sp. 2. Staphylococcus epidermidis3. Enterococcus faecalis 4. Pseudomonas aeruginosa5. Escherichia coli6. Serratia marcescens

Procedure:

1. Your group will make one agar plate for each of the stock culture. You will use nutrient agar plates for all of the cultures. Before you inoculate, label the bottom of each plate (along the edges) with your table number, name of the bacteria you will use, and the date.

2. Flame the inoculating loop and allow it to cool. Remove the cap of the stock culture and flame the opening.

3. Insert the loop into the tube and pull it out slowly. There should be a film of broth inside the loop. If it pops, try again.


4. Re-flame the mouth of the tube and recap it. Return it to the rack – you might want a partner to help you with this as you are carrying a loop filled with a bacterial culture.

5. Carefully lift of the lid of the plate. Do NOT place the lid with the rim facing down on your working surface. This will contaminate your lid. If you need to set your lid down, set your lid down, rim facing up.

6. With your loop, gently mark a line on the agar, from the top of the agar plate moving down the midline, approximately 1 cm. This will note the place that you initially deposited your culture. Be careful not to gouge the surface! (This is crucial!)

7. Place the loop outside of the starting midline to the left and move the loop across the midline to the right. Continue streaking for about 1/3 of the plate. See image above.

8. Rotate the plate a 1/3 turn. Flame and sterilize your loop. Place the loop outside the streaked area. Move through the streaked area one time then continue streaking away from the first area. Streak for 1/3 of the plate, being careful not to cross (move) the loop through the original streak.


Individual colony

Variation in streaking protocol: It is not always necessary to

flame and sterilize the loop after each 1/3 turn of the plate, particularly if a known species of bacteria grows small punctiform or circular colonies.

If colonies tend to be large or if overgrowth of medium is a concern, it is best to flame the loop after every turn.

9. Rotate another 1/3 of a turn, flame your loop, and repeat. This will create individual colonies since you are essentially spreading out the bacteria and isolating one bacterium from another.

10. Replace the lid, re-flame the loop and let it cool.

11. Tape the lid shut. You will be incubating the inoculated plates, lid-side down, for 24-48 hours. Note the incubation temperature of 37 degrees Celsius in your lab manual.


Lab 3: Observation of Cultures & Colony Morphology

Purpose: To practice observing broth and agar cultures for evidence of growth, as well as to learn to classify colonies by specific descriptors.

Background:Bacteria have exceedingly high rates of binary fission and are therefore easy to culture in a lab. Evidence of growth in a broth medium will be seen as a cloudy appearance in a previously transparent solution. The bacteria will tend to form a pellicle at the bottom of the tube and can be suspended by vortexing or careful mixing.

Agar colonies start with only one bacterium! Each dot on the agar is a colony consisting of thousands or millions of clones of the original bacterium. Each species produces colonies that have characteristics specific to that species. By careful observation, you should be able to differentiate between individual colonies.

Procedure A: Observation of Broth Cultures1. Examine each tube from Lab 1 for evidence of growth. Evidence of growth would appear as cloudiness (known as turbidity), a collection of material at the bottom of the tube (a pellicle) or along the top of the tube. A tube that has no growth would remain clear and transparent.

2. Create a table for your results. Make sure to note the organism that was inoculated in broth and the date. Note the results and color of the culture of both the control and the experimental tube in your lab notebook. Make necessary observations. Be clear in your data, observations, and drawings. Don’t forget to get a witness for your data!

Procedure B: Observation of Streaked plates1. Maintain careful records of the observations of each streaked plate. You need to be organized with your records and detailed in description. Create an appropriate data table to accomplish this.

2. Observe the plates looking for individual colonies. These spots should be isolated from each other – not touching.

3. Use the following descriptors in your data table to record in detail about the appearance of the colonies so that you can use this information when you work with your unknown, mixed cultures. Include an extra column in your data table for remarks or additional observations that you may want to note about each species of bacterium.


Descriptors of Colonies:

A. SIZE OF COLONY (measure with a millimeter rule), less than 1mm = punctiform (pin-point).

B. EDGE/MARGIN OF COLONY: See image below

C. COLOR: white, buff, red, purple, etc.

D. OPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass), iridescent (changing colors in reflected light)

E. ELEVATION OF COLONY (turn the place on end to determine height)

F. SURFACE OF COLONY: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)

G. CONSISTENCY: buttery, viscid (sticks to loop, hard to get off), brittle (dry, breaks apart), mucousy

H. ODOR: Absent or present? If it has an odor, what does it smell like?

**Some characteristics are easier to see and note than others. If a characteristic cannot be determined, do not make-up data. Simply mark your data table accordingly. For example, “not discernible”, or ND.


Lab 4: Bacterial Staining Methods

Purpose: To learn methods to stain bacteria which are almost colorless and therefore show little contrast with the broth in which they are suspended. Using gram staining, cultures can be differentiated based on their shape, arrangement and cell wall structure.

Materials for each lab group:Lab stove/Bunsen burnerSharpieInoculating loopAgar plates from Lab 2 with the following bacterial cultures:

Bacillus sp. Staphylococcus epidermidis Enterococcus faecalis Pseudomonas aeruginosa

Escherichia coli

Serratia marcescens

GlovesGram’s Iodine Crystal VioletSafraninDecolorizer Staining trayForcepsPaper towel or bibulous paperMicroscope slidesOil Immersion microscopeImmersion oilOil Immersion CleanerQuality Control (QC) slide

Background for Heat Fixation:Before staining bacteria, you must first understand how to "fix" the organisms to the glass slide. If the preparation is not fixed, the organisms will be washed off the slide during staining. A simple method is that of air drying and heat fixing. The organisms are heat fixed by passing an air-dried smear of the organisms through the flame of a gas burner. The heat coagulates the organisms' proteins causing the bacteria to stick to the slide, killing the bacteria in the process.

Background for Staining:In order to understand how staining works, it will be helpful to know a little about the physical and chemical nature of stains. Stains are generally salts in which one of the ions is colored. (A salt is a compound composed of a positively charged ion and a negatively charged ion.) For example, the dye methylene blue is actually the salt methylene blue chloride, which will dissociate or dissolve in water into a positively charged methylene blue ion which is blue in color and a negatively charged chloride ion which is colorless.

Dyes or stains may be divided into two groups: basic and acidic. If the color portion of the dye resides in the positive ion, it is called a basic dye (examples: methylene blue, crystal violet, safranin). If the color portion is in the negatively charged ion, it is called an acidic dye (examples: nigrosin, congo red).


Because of its chemical nature, the cytoplasm of all bacterial cells has a slight negative charge when growing in a medium of near neutral pH. Therefore, when using a basic dye, the positively charged color portion of the stain combines with the negatively charged bacterial cytoplasm (opposite charges attract) and the organism becomes directly stained.

The Gram stain was developed in 1884 by a Danish physician Hans Christian Gram while working in a morgue in Berlin. He was not satisfied with his stain; however, because not all of the bacteria seemed to retain the stain equally. What he considered a defect in his staining technique turned out to be the basis of the most widely used test for distinguishing bacteria from one another!

Bacteria are either gram-positive and stain purple, or gram-negative, and stain reddish-pink. The distinction between them is based on the type of cell wall they have. Gram-positive bacteria have a thick outer cell wall made up of peptidoglycan, a protein-carbohydrate complex. Gram-negative bacteria also have a peptidoglycan layer but it is much thinner, and the peptidoglycan is sandwiched between two plasma membranes of phospholipids. The outer membrane is rich with fats and sugars. Because their cell walls have different biochemical make-ups, they accept dyes in different ways. A differential stain is one in which different dyes are present in the stain that will differentiate types of cells.

The gram stain requires four solutions: a purple dye called crystal violet, a mordant, a decolorizing agent and a counterstain. A mordant is a substance which causes the stain to stick to the cell more strongly, that is, it “fixes” the dye to the bacterial cell. The mordant used in the Gram stain is Gram’s iodine solution.

A decolorizing agent removes the first dye from the stained cell. In the Gram stain procedure the gram negative cells will have the crystal violet dye removed more quickly because their peptidoglycan layer is thinner. The decolorizing agent we are using is a 50/50 mixture of 95% alcohol and acetone. Alcohol is an organic solvent and will dissolve the crystal violet out of the cell wall of a gram negative organism. This is the critical step in


the gram stain procedure; leaving the alcohol on too long can begin to decolorize the gram-positive bacteria.

The counterstain is a dye of a different color from the original dye. The cells that are decolorized will take up the color of the second stain. The counterstain used in the gram stain is called safranin and is red.

The cells that retain the crystal violet are purple and are called gram-positive; the cells that are decolorized with alcohol and counterstained to red are called gram-negative. It is important to know if a bacterium is gram-positive or gram-negative because of the differences in cell wall structure. It is also significant since antibiotics are absorbed differently with gram-positive and gram-negative bacteria. As a result, treatments for infections from gram-negative organisms require different antibiotics than for gram-positive bacteria. Also, the cell wall of gram-negative bacteria is very toxic to many animals and infections with gram-negative bacteria tend to be more serious.

Procedure 4A: Heat Fixation of Bacteria onto Slides1. Using a pencil, label 6 slides (on the frosted end of the slide) with the name of the each

of the stock bacteria.

2. Aseptically remove a small amount of the culture from the agar surface and smear it on a labeled slide near the frosted end of the slide. Use one slide per organism. You may also use a sterile swab to pick the organism from the agar and smear on the slide.

3. Burn the remaining bacteria off of the loop.

4. Pass the slide (film-side up) through the flame of the bunsen burner 3 or 4 times to heat fix.

Procedure 4B: Gram Staining1. Place the slide on a staining tray and cover the entire film with Crystal violet. Leave the

stain for one minute.

2. With a gloved hand, pick up the slide by one end and hold it at an angle over the staining tray. Using running tap water, rinse off the crystal violet with a gentle stream of water. Also wash off any stain that got on the bottom of the slide as well.

3. Cover the slide with Gram iodine solution for 1 minute. Again, rinse with gentle tap water.

4. Holding the slide, gently drip the decolorizer on the slide. Watch as the crystal violet is washed from the smear. Stop decolorizing as soon as the purple stops flowing from your smear. This might only be 1 or 2 drops! Watch carefully as it is possible to over-decolorize your smear! Immediately rinse the slide in the gently running tap water.

5. Place slide in staining tray and cover slide with safranin counterstain for 1 minute.


6. Gently rinse with water and VERY CAREFULLY blot dry.

7. Follow the same procedure to gram stain the QC – Quality Control slide. The purpose of the QC slide is to act as your control, to make sure you effectively gram-stained your experimental slides.

8. Set aside for viewing during the next lab. Note that each member of your group will need to examine every culture because each of you will be assessed individually on your lab notebook.


Lab 5: Bacterial Morphology Lab

Purpose: To verify proper gram staining process using the QC slides and to develop an accurate description of each species’ morphology and Gram reaction. This information can be used to later help in the identification process of each species of bacterium.

Background:There are three basic bacterial shapes:

a. round or cocci (coccus singular)b. rod-shaped or bacilli (bacillus, singular)c. spiral-shaped or spirilla (spirillum, singular)d. comma-shaped (curved rod) or vibrios (vibrio, singular)

Bacteria can be found in different arrangements:a. singly – as one single bacteria not adhering to any othersb. diplo – a pair of bacteria joined together (diplococcus)c. strepto – a chain of bacteria (streptococcus)d. staphylo – a grape like cluster of bacteria (staphylococcus)

Bacteria will have characteristic Gram stain reactions:a. Gram positive: maintained a purple colorb. Gram negative: were decolorized and appear red to pink

By putting the shape and arrangement together, the morphology of the bacteria can be described. For example, if the bacterium is round and found in a chain, it is streptococcus. If it is a rod and found in pairs, it is diplobacillus. The bacteria can be further described by their staining reaction with the Gram stain. (i.e., gram positive staphylococci or gram-negative bacilli.)

It is important to note that the arrangement of the bacteria cannot be determined if the bacteria used for fixing and gram staining came from an agar medium. The solidity of the agar medium tends to restrict the natural arrangement of the bacteria. The bacteria will only form arrangements if they were fixed onto the glass slide from a broth suspension.


Reading the QC Slide to Verify Proper Staining ProcessIf you performed the staining process correctly, your QC slide will show gram positive (purple) cocci and gram negative (red) rods. If you see red cocci or purple rods, the staining process was incorrect, and this will indicate that the results of your stock culture stains may also be incorrect.

Materials:Gram Stained slides from Lab 4Microscope with oil immersion lensImmersion oil TIPS FOR MICROSCOPIC OBSERVATIONS

1. Remember that in the process of making the slide, some of the coccal arrangements will clump together and others will break apart. Move the slide around until you see an area representing the true arrangement of each organism.

2. Small bacilli (such as Escherichia coli) that have just divided by binary fission will look similar to cocci. Look carefully for bacilli that are not dividing and are definitely rod-shaped as well as bacilli in the process of dividing to confirm the true shape.

3. Bacilli do not divide so as to form clusters. Any such clusters you see are artifacts from preparing the slide.

Procedure1. Always use the lower power objectives to initially focus and find bacteria. But to

view them, you must use the 100X oil immersion lens.

2. Place a drop of oil on the slide over the area to be examined (cover slip is not needed), then carefully slide the oil immersion lens in place. The objective will adhere to the oil droplet and form a continuous column.

3. Carefully focus and find the bacteria, adjusting light as needed.

4. Keep detailed records of your observations in your lab notebook: draw the bacteria true to color. In your annotations, note the shape and Gram stain reaction. Your drawings will serve as your primary data. Don’t forget to follow all rules for dealing with data. (i.e., title, magnification, annotations, labels, etc.) Do not forget to get a witness for your data.

5. If you have a camera, on your phone, for example, you have an additional option of photographing your images. However, this is your secondary data source, as you are probably not equipped with proper microscope cameras, and the images may be distorted and not true to color. You may also loose resolution in your images. If you choose to include photographs, you will still need to follow rules for dealing with data, as listed above.

6. When you are done with the scopes, the oil immersion lens and the stage need to be


carefully cleaned with lens paper and lens cleaner.

Lab 6: Differential vs. Selective Media

Purpose: To discover how different types of culture media can aid in the selection, isolation and identification of bacteria.

Background:To study a particular influence on a pure culture in the laboratory, biologists alter one variable at a time. But in nature, many variables are influencing microbes at any one time, and their overlapping interactions determine the fate of the organisms.

One goal in studying microbiology is to understand how the microorganism functions in its habitat. In a practical sense, the ultimate control of microorganisms in medicine, food industries, and environmental microbiology depends on this understanding. An understanding of how a microorganism lives is required before steps can be taken to inhibit or kill it.

Habitats rarely contain only a single species of microbe. To determine what microbes live in a habitat, every species present must be isolated as a pure culture and identified. Sometimes this may be done by testing for particular characteristics of the organisms, i.e., their salt tolerance or temperature range. For example, a halophilic organism can be separated from other bacteria in a sample by growing the culture on high salt media. The microbes that can’t live in this environment won’t grow. This is known as selective plating. Something in the medium selects the organism that is of interest over the other bacteria in the sample.

Sometimes a microbe can be isolated or identified by making it easier to recognize among the other bacteria in the medium. It doesn’t necessarily inhibit the other organisms; it simply makes the bacteria of interest stand out from the others. This is known as differential plating.

The human intestinal tract contains over a billion bacteria of many different species. Humans are in a symbiotic relationship with most of these bacteria; they live off of the undigested food in our waste, further digesting it. In addition to aiding in digestion, they also produce essential vitamins as part of their waste products. These bacteria are known as enteric (enter/o = small intestine) bacteria. Unfortunately, if they get into parts of the body where they don’t belong, such as the abdominal cavity or the urinary tract, they can cause disease. There are also some bacteria that cause disease in the intestinal tract and are not found their normally, such as Salmonella sp. When culturing samples that are contaminated with enteric bacteria, we need a way of distinguishing them from one another.


EMB Agar Background:Eosin-methylene blue (EMB) agar is both a selective and a differential medium for enteric bacteria. It is selective because gram-positive organisms will not be able to grow on the agar at all. The aniline dyes (eosin and methylene blue) inhibit the growth of gram-positive organisms by some unknown mechanism. It is differential because colonies of E. coli will turn black on eosin-methylene blue agar (EMB), while other organisms will be clear or pink on the same agar.

Some bacteria use the lactose for food, giving off acid waste products which are detected by the pH indicator eosin. Lactose fermenters (Lac + bacterium) will drop the pH of the agar. The acid changes the color of the agar and the color of the colony. They will typically appear black or have dark centers. Non-lactose fermenters (Lac – bacterium) will make clear or opaque, light pink, or in general, lighter-colored colonies on the EMB agar. Strong acid-producing bacteria, such as E. coli, form colonies that have a green metallic sheen. (This metallic sheen is unique to E-coli only!)

Using the appearance of the colonies on EMB, a presumptive identification can be made and then they can be selected, sub-cultured for purity and studied further. For example, if a urine specimen from a patient suspected of having a urinary tract infection (UTI) is being studied, the black colonies of E. coli, the most common cause of UTI would be sought. However, if culturing a stool specimen from someone suspected of being infected with Salmonella, black colonies would be ignored, and clear colonies would be looked for, because Salmonella doesn’t use lactose as an energy source.

CNA Agar Background:Colistin Nalidixic Acid agar, known as Columbia CNA, is a selective and differential media used for isolating gram positive organisms. Colistin and Nalidixic acid are antibiotics that inhibit the growth of gram negative bacteria. This media also contains sheep blood which gives it a red color. The blood in the agar supports the growth of many gram positive organisms such as staphylococcus and streptococcus. Staphylococci can cause significant skin infections and streptococci can cause strep throat. Some of the gram positive organisms produce an enzyme that will lyse (hemolyze) the blood in the agar and leave a clear area around the colony.

Typically, there are three categories of hemolysis. Alpha:• Hemoglobin is converted to methemoglobin in the medium surrounding the colony. • This produces a green discoloration of the medium.

Beta:• Lysis of RBC• Yellow or clear zone surrounding the colony

Gamma:• No hemolysis. • No destruction of RBC• No change in medium


MacConkey Agar Background:MacConkey agar is a selective and differential plating medium for the selection and recovery of the Enterobacteriaceae and related enteric gram-negative bacilli. The agar contains bile salts, crystal violet, lactose carbohydrate, and a pH indicator called neutral red. The bile salts and crystal violet in the agar serves to inhibit the growth of gram-positive bacteria and some slow growing gram-negative bacteria; therefore, this medium will promote the growth of gram-negative bacterium. The lactose, allows differentiation of these gram-negative bacteria based on their ability to ferment lactose. Organisms which ferment lactose produce acid end-products which react with the pH indicator neutral red, and produce varying shades of pink in their colonies.

Typical strong lactose fermenters, such as Escherichia coli, produce dark pink colonies surrounded by a zone of precipitated bile. Slow or weak lactose fermenters, such as Serratia marcescens, may appear colorless after 24 hours or slightly pink in 24-48 hours. Colonies of non-lactose-fermenting bacteria, such as Pseudomonas aeruginosa, appear colorless or transparent.

Using these three agars, it is possible to separate gram positive and gram negative bacteria in mixed cultures and also determine if the bacterium has the ability to ferment lactose as a carbohydrate source. It is also possible to determine a possible identification based on the appearance of the colony on different media.

Materials:The six stock cultures:

1. Bacillus sp. 2. Staphylococcus epidermidis3. Enterococcus faecalis 4. Pseudomonas aeruginosa

5. Escherichia coli

6. Serratia marcescens 3 EMB agar plates3 CNA agar plates3 MacConkey agar plates1 nutrient agar platesInoculating loopsLab stoveSharpie pens

Procedure:1. Using a marking pen, divide the bottom of each EMB, CNA, and MacConkey agar

plates into two equal sections. Mark with your table or group, initials, date, and label each half with the appropriate bacteria.

2. Inoculate each of the stock cultures on one half of an EMB, CNA, and MacConkey agar plates. Try to streak for isolation and remember to use aseptic technique. You may want to flame your loop after each turn of the plate, since you have less surface area for inoculation, and you want to insure isolation of colonies. Follow the diagram on the following page for proper streaking pattern on a half plates.

3. Incubate the plates upside down at 37° for 24 hours. Note the temperature and incubation length in your notebook. Refrigerate plates over the weekend.


4. After 24 hours, observe the EMB, CNA, and MacConkey plates for growth of each type of bacteria. Carefully note the response of each culture to each plate type. Record growth, colony color and appearance. Look for the presence or absence of hemolysis on the CNA plate. Look for evidence of lactose fermentation on the EMB and MacConkey Agar plates.

5. If a camera is available, take a photograph of each of your plates.

6. Deal with all data appropriately and get a witness.

7. If you suspect any errors in technique were made, make a note of that in your notebook.

Results:

Create a chart similar to this for your data. You and your teammates may design a table that is different from this. Make sure you draw your data table large enough so that you can properly record observations. It is suggested that you use an entire page for your data table, and that you create your data table in landscape. Don’t forget to use a straight edge! No free-hand.

EMB Agar CNA Agar MacConkey Agar Other Observation

sOrganism Growth

orNo growth

Color of Colonies

Lac+ or Lac-

Growth orNo growth

Color of Colonies

Hemolysis and type?

Growth or No growth

Color of Colonies

Lac + or Lac -


Attach any photographs of the plates into your notebook. Make sure to title, label, annotate, witness, etc.

Lab 7: Carbohydrate Fermentation

Purpose: To determine the differing nutritional requirements of bacterium and to use this information to differentiate them.

Background:Most organisms, including humans, need a source of glucose for energy. Cells are able to break down glucose because of their various enzymatic systems. Each bacterial species has different combination of enzymatic systems and therefore are able to utilize different niches in nature, lessening competition. Organisms are adapted to the environment in which they live and are able to take advantage of the food sources available. Some organisms have enzymes that can convert lactose and sucrose (both disaccharides) into glucose to utilize them for energy. As they break down the carbohydrates, bacteria will release a waste product – often lactic acid and carbon dioxide. Most bacteria ferment their energy sources, even though they live in an atmosphere high in oxygen. Some bacteria however are oxidative, and the pH change will only be visible at the top of the tube where oxygen is available for metabolism.

The pH indicator being used is phenol red, which is added to the broth containing a particular sugar. It is red at pH 8.5 and yellow at pH 6.9. It will change color as the acid is produced by the fermenting bacteria. A Durham tube, which looks like a miniature test tube, is inserted upside down in the broth culture and will catch carbon dioxide bubbles as they are produced.

MaterialsLab stoveInoculating loopMarking pen and lab tapeTest tube rackThree tubes of each of the following phenol-red broths per group:

3 PR broth with glucose + Durham tube 3 PR broth with lactose + Durham tube

Assigned cultures – each group will test three organisms:

Lab Stations 1, 3, 5, and 7 Lab Stations 2, 4, 6, 81. Bacillus sp. 2. Staphylococcus epidermidis3. Enterococcus faecalis

4. Pseudomonas aeruginosa5. Escherichia coli6. Serratia marcescens


Procedure

1. Each group will be responsible for testing three different organisms in each of the broth solutions. Label each tube of broth with your group name, date, and the bacterial culture being tested.

2. Aseptically transfer some bacteria from the stock plate to the glucose broth tube – being careful to flame the opening of the PR broth tube before recapping it. Place in test tube rack.

3. Repeat for the lactose broth.

4. Repeat the procedures 2 and 3 above for the other two assigned cultures, aseptically transferring each culture onto both the glucose and lactose broths.

5. Incubate all tubes for 24 hours at 37 °.

6. Come in the next day and accurately record results. All groups will need to share data at the next lab session.

Results:Create a chart similar to the one below and use the suggested abbreviations. Gather data from other groups for each of the cultures. A = acid produced – broth changed from red to yellowG = gas produced – bubbles present (or obvious displacement of solution) in Durham tubeA/G = acid and gas are both producedNC = no change in color, no gas present * Make sure to note the color change, if any.**Also describe if the change in color extends throughout the tube or is only present at the top of the solution.

Organism PR/Glucose + color PR/Lactose + color Conclusions about culture


You will share your data with your classmates to complete the data table. Since each organism will be tested three times, consider outliers before making any conclusions about each culture’s fermentation properties.

Lab 8: Biochemical Tests

Purpose:In addition to carbohydrate fermentation, there are many other tests that can be done on bacteria to distinguish them from one another. The results of the tests usually depend on whether the organisms contain a particular enzyme to break down the substance being used or whether they can use it as a source of nutrition. Different organisms in the laboratory can be identified utilizing the following biochemical tests.

Advanced Student Prep: In order to perform biochemical tests, the organisms need to be freshly cultured. 24 hours prior to this lab, you will need to inoculate 3 nutrient agar plates and 3 starch agar plates with your 6 species of bacterium during FIRE. Divide the bottom of each plate into two equal sections, and aseptically transfer pure colonies onto each ½ of the plate. Label each side with your table number, date, and name of organism. Incubate plates for 24 hours at 37 °.

Data Table Construction:Create a data table similar to the one below prior to conducting the biochemical tests:

Organism Starch hydrolysis

Catalase Test

OxidaseTest

IndoleTest

Additional Observations

Record results as positive or negative, or describe the appearance of the tube or plate. Be sure to leave enough room to make additional detailed notes on the appearance of each test.


Test #1: Starch Hydrolysis Procedure 1A

Background:Starch is a complex carbohydrate that some bacteria can use for energy, but only if they produce the enzyme amylase, which breaks down starch into glucose. A test for the presence of starch is the appearance of a blue-black color upon the addition of iodine. If an organism contains amylase, the starch will be broken down into glucose and the blue color will NOT be produced. A clear zone around the colonies of bacteria indicates the presence of amylase; a blue color indicates that the bacteria do not produce amylase.

Materials:Bunsen burnerIodine solution3 Starch agar plates6 Stock cultures

Procedure:1. Using a marking pen, divide the bottom of a starch agar plate into two equal

sections. Mark with your group, date, and label each half with the appropriate bacteria.

2. Use sterile technique to inoculate the plate with E.coli and Bacillus sp. by making a straight line streak down each side of the plate.

3. Do the same with the four other organisms – one streak on each side of a plate.

4. Incubate the plates upside down at 37° for 24 hours.

Test #1: Starch Hydrolysis Procedure 1B

1. After a 24-hour incubation, flood the plate with iodine solution and look for a clear zone around the line of bacteria.

2. Record results and what the color, upon the exposure to iodine indicates.


Test # 2: Catalase Test

Background:Catalase is an enzyme that is produced by most aerobic bacteria. It catalyzes the breakdown of hydrogen peroxide to water and oxygen.

When 3% hydrogen peroxide is added to a slide preparation of bacteria, it will bubble if catalase is present. The bubbles indicate the production of oxygen gas. If there are no bubbles, the organism is catalase-negative. Catalase-negative organisms tend to be anaerobic and include the genera Streptococus, Lactobacillus, and Clostridium (the bacteria that causes botulism)

MaterialsLab stoveHydrogen peroxide, 3%Inoculating loopMicroscope slidesStock cultures

Procedure1. Obtain six clean microscope slides.

2. One at a time, use a lab stove and aseptic technique to make a visible smear of each of the 6 stock organisms a slide.

3. Put a drop of 3% hydrogen peroxide on top of the organism smear. Watch for the formation of bubbles.

4. Record the results.


Test # 3: Oxidase Test

Background:The cytochrome oxidase test uses certain dyes, such as p-phenylenediamine dihydrochloride. The dye is colorless; however, in the presence of the enzyme cytochrome oxidase and atmospheric oxygen, p-phenylenediamine is oxidized, forming indophenol blue. Pseudomonas sp. are positive for oxidase, and enteric gram negative bacilli, such as E. coli, are negative for oxidase.

MaterialsLab stoveFilter paperSterile swabs or sterile plastic loops (do not use metal loops) Stainless steel or Nichrome inoculating loops or wires should not be used for this test

because surface oxidation products formed when flame sterilizing may result in false-positive reactions.

Oxidase reagentStock cultures

Procedure1. Obtain a piece of filter paper. Place a drop of oxidase reagent on the filter paper.

2. Pick up a sample of bacteria from a stock culture with the wooden end of a sterile swab. Rub the bacteria onto the filter paper.

3. If the bacteria turn black or purple within 10 seconds, the bacteria utilize the enzyme cytochrome oxidase and are considered oxidase positive. If it remains the same color, it is negative for oxidase.

4. Do not record results after 10 seconds, as the results after 10 seconds will give you false readings.


Test # 4: Indole Test

Background:This test is used to determine the presence of the enzyme tryptophanase. Tryptophanase breaks down the amino acid tryptophan to release indole, which is detected by its ability to combine with certain aldehydes to form a colored compound. Indole positive organisms produce a blue-green compound formed by the reaction of indole with cinnamaldehyde. Indole negative organisms lack the enzyme tryptophanase and produce no blue green color.

MaterialsLab stoveFilter paperInoculating loopIndole reagentStock cultures

Procedure1. Obtain a piece of filter paper and add one drop of the 1% Indole reagent.

2. Flame an inoculating loop and pick up a sample of bacteria from a stock culture. Rub the loop of culture onto the filter paper.

3. Rapid development of a blue-green color indicates a positive test. Most indole-positive organisms develop color within 30 seconds. A reddish color may be produced by an indole negative organism.


Lab 9: Identifying a Mystery Culture

Purpose: To apply systematic procedures to identify two unknown bacteria in a mixed culture.

MaterialsNutrient, EMB, CNA, MacConkey and Starch agar platesGram stain reagents3% Hydrogen peroxide (catalase reagent)Oxidase reagentIndole reagentMicroscopes and slidesMarking pen, lab tapeSaline suspension with three unknown bacteria

Procedure:1. Examine results from all of the completed experiments thus far in this course. Create a

dichotomist key to organize this information. You will use your dichotomist key to help identify your mystery culture. Below is an example of how to create a dichotomist key.

2. Create a master table that consolidates all the data that you’ve compiled in labs 1 – 7 and transfer the data.

Medical Technology Microbiology Lab Manual Page 30

3. Design a series of tests and procedures to separate and identify the three different types of bacteria in the provided saline suspension. Write this up as a flow chart and get approval before beginning.

4. Plate your saline suspension of organisms on Nutrient Agar, EMB, CNA, and MacConkey Remember to streak for isolation. Be sure to note the number of your unknown. Carefully label each plate with your group name, unknown number and date of inoculation.

5. After obtaining isolated colonies on your selective media, streak one colony of each type onto a nutrient agar plate to obtain a pure colony to be used in biochemical testing. You may need to re-plate the organisms more than once to obtain a plate of a pure organism. You may then work to identify the cultures with at least two supporting biochemical tests plus gram stain. Your support must be incontestable. Be sure to differentiate your unknown bacteria with labels such as 1,2,3 or A,B,C.

6. Start biochemical tests as necessary to make definitive identification of your cultures. If you will need more than one day to do your testing, make sure to re-plate the organisms to fresh nutrient agar plates. Biochemical testing should be done on plates no more than 48 hours old.

7. Keep organized and detailed records throughout your experiment.

Conclusion:Write up a formal statement with your lab group, identifying your unknown cultures that will be typed up and turned in with your lab notebook for final assessment. Directions are on the following pages 32 – 34, with the grading rubric for both the paper and your notebook that follows.

Good luck!!


Mystery Culture Scientific Paper

This will be a formal statement identifying your unknown cultures that will be typed and turned in. This will be a “group” paper. Researchers that work on a project publish one paper, not several individual ones. So divide the work equally and conquer. Peer-edit each other’s sections so everyone is happy, then submit this paper for grading, along with each individual group member’s lab notebook.

Voice: All sentences must be in the present tense, except for “methods”, which are in past

tense. Do NOT use 1st person personal pronouns. The experiment should always be the

subject of your sentences, NOT yourselves! (For example, do NOT say, “We used the streak-plate method.” Instead say, “The streak-plate method was used to separate bacteria from each other.)

Formal writing. No Humor or slang.

Format: Required Length: 10-14 pages 12 point font: standard font only. Line Spacing: 1.5 Abstract will be centered on top of the page. Use double column format for most of the paper (Introduction, Methods, and

Conclusion) Highlight the sections that need to be in columns. Then, go to Page LayoutColumnsand choose 2 columns

Tables, Graphs, and Pictures do not need to be in columns. However, all data must be properly titled, labeled, annotated, etc.

Add a footer to number your pages Cover page: Include your authors’ names, date, class, teacher, and period

somewhere on your cover.

Tips for Formatting:Group members:

Do not format any part of your paper. Send your entire work to the editor, unformatted without columns, page numbers, footers, etc.

To the Editor: Consolidate all the written work from your group first. Check it for errors. Make sure that no information is being repeated and that all information is in the

right sections. (No methods in background, for example). Then format the entire document at the end and add columns, page numbers,

footers, and use consistent font.


Order and Subject of Paragraphs:

I. Abstract: (Include this title in your paper and center it!) ~ 1 paragraph

An abstract is a one paragraph (6-10 sentences) summary of your entire project. You must use the IMRaC format for writing an abstract. In other words, you use one to three sentences to describe each of the following main points about the experiment (not about the classroom procedures) in your one-paragraph abstract:

I= Introduction (purpose)M= Methods (simple overview of procedure)R= Results (numerical data)

andC= Conclusion (interpretation of data)

II. Introduction: (Include this title in your paper)~3-4 pages in columns

This sets the stage for your experiment. Introduce the lab and purpose of the lab. Explain the significance or importance of the experiment.

Use cited research to provide some background on bacteria as well as background on the lab procedures. Research should include the following topics:

o General overview of bacteria: general prokaryotic propertieso Explain bacterial classification: shapes, arrangements, nutritional

requirements (carbohydrate fermentation), gram + vs gram – cell wallso Brief explanation of each of the following tests used to identify the unknown:

gram staining, all differential vs. selective medias, carbohydrate fermentation in durham tubes, various biochemical tests. Do not write about the methods here. This is just an explanation of the scientific background behind each test. If in doubt, refer to the background given in your lab manual.

State the problem/question- The research you provide needs to lead into the question that is being investigated in this experiment. Make sure your scientific question is worded clearly.

III. Methods: (Include this title)~2-3 pages in columns

Summariz e the methods (procedure) used to conduct your experiment in paragraph form. The following methods should be included:

o Aseptic Techniqueo Streak Plate Method for Isolation on each type of agar.o Gram staining and Morphology Identification o Carbohydrate Fermentationo All Biochemical Tests

This should be a description of the work flow that describes the laboratory tests used to identify the unknown culture. This is NOT a list of materials or equipment! Obvious steps, like recording data or labeling tubes, do not need to be mentioned.


DO NOT number the steps of the procedures and do NOT copy the procedure section out of your book!

Explain what was established as your quality control group, and what was established as your experimental groups throughout the various tests.

IV. Results: (Include this title)~1-2 pages (This section does not need to be in columns)

Include the dichotomist key that you created of your original six cultures. Include the “master” table. This would be your “known” data. Choose the most appropriate method for representing your “experimental” data.

Display your results in words, figures, tables, and pictures. Tables must be computer generated and may not be done by hand! Deal with data appropriately. All data needs to be titled, labeled, magnification

indicated when appropriate, annotated, etc. All data must be supported with annotations to explain its significance. All images must be digitally inserted, not cut and pasted. Label all graphs: For example, Figure 1, Figure 2, Table 1, etc. Tips: Do NOT break a table up into different pages. Make sure your table fits on one

page. If it doesn’t, start the table on the following page.

V. Discussion/Conclusion (Include this title) ~3-4 pages in columns

First, restate the original problem and original purpose Conclude the identities of your three bacterial species. Discuss how and why you determined the identity of your bacteria. Use the data to

help explain and support the identities of your bacterium. Be sure to describe how the tests provided information on the identities of your unknowns. Explain the process used to compare your unknown bacteria to the known characteristics of your original six bacterial cultures.

Explain any errors or problems that you had with the identification process of the bacterium. Consider the following: Did you have to repeat any tests? Did you get isolation the first time or did you have to re-streak? Did you ever get inconsistent results? Was there any source of contamination? You are not limited to these questions. Address any problems with the experiment here. Explain if the results were affected, if at all.

Do not use the word PROVE in a conclusion. Very few phenomena can be proven. Application: Discuss ways that improvements can be made. If you have trouble with

this, think of times when you had errors or problems with the procedures. How would these problems be avoided in you had to repeat this experiment?

Discuss the value of this data. How is this information useful? How would this information be applied to the field of medicine or perhaps other field of study? How would the general public or scientific community use this information?


Scientific Paper Scoring Rubric for Mystery Culture: Medical Technology

Section 1: Manuscript Requirements (5 points) Superior Good Adequate Inadequate None

Typed

All titles present: Abstract, Introduction, Methods, Results, Conclusion

Cover Page w/ name, date, class, period, and teacher. (option extra credit magazine cover ). If extra credit option is chosen, place cover page behind the magazine cover but in front of paper.

10-12 point standard font, 1 inch margins, 1.5 line spacing, page numberings

Abstract and Results ~ single columnIntroduction, Methods, Conclusion ~ double columnsTotal Points for Manuscript : _____/5Section 2: Abstract (10 points) Superior Good Adequate Inadequate None

1–3 sentence introduction (present tense, 3rd person)

1-3 sentence methods (past tense, 3rd person)

1-3 sentence results (present tense, 3rd person)

1-3 sentence conclusion (present tense, 3rd person)

Concise, well-written paragraph summarizes the entire experiment. Meets conventions of English and is error-free.

Total Points for Abstract : _____/10Section 3: Introduction (20 points) Superior Good Adequate Inadequate None

Introduces the lab and purpose/significance of experiment (3)Provides researched background on bacteria and necessary information on laboratory tests that are to be used for identification of unknown culture (must include in-text citations of all scientific information and graphics that are not considered “common knowledge” to the general public ~ lack of citations will be considered plagiarized) (10)Background information leads into the statement of the investigative problem (investigative question) (2)

Meets length requirement; well written, follows conventions of English, and organized into paragraphs; Error free. Present tense, 3rd person (5)

Total Points for Introduction: _____/20Section 4: Methods (15 points) Superior Good Adequate Inadequate None

Procedure summarized such that another scientist reading the procedure can duplicate the experiment exactly. All procedures present. None are missing (8)

Properly identifies control and experimental groups (2)Meets length requirement; well written, follows conventions of English, and organized into paragraphs. Error Free. Past tense, 3rd person. No numbering of procedures. (5)Total Points for Methods: _____/15Section 5: Results (15 points) Superior Good Adequate Inadequate None

Chose an appropriate method for organizing data; display data in words, figures, tables, and/or pictures.

All tables, graphs, figures or pictures are computer generated, neat, and complete with accurate information.

Tables, graphs and pictures are titled. Data submitted must be relevant to the identification of your culture.

An informative 1-2 sentence caption is provided for each table, graph, or picture; in present tense, 3rd person

Meets length requirement of 1 – 2 pages; follows conventions of English, and organized into paragraphs. Error free. Present tense, 3rd person.

Total Points for Results: _____/15



Section 6: Conclusion (20 points) Superior Good Adequate Inadequate None

Problem (investigative question) are restated (1)Identifies the unknown bacterium in the mixed culture using results to support their conclusion. Results are well described and interpreted, using background knowledge of the known cultures compared to the unknowns. Infers an explanation for the results based on knowledge of science concepts. The word “PROVE” is not used. (8)

Identifies sources of error, and how results were affected. Explains how procedures can be modified, based on these errors. (4)States how the findings of this lab can be applied or be useful to other fields. Proposes new experiment or gives suggestions for further study. (2)Meets length requirement; follows conventions of English, and organized into paragraphs. Error free. Present tense, 3rd person. (5)Total Points for Conclusion: _____/20Section 7: Technical and Objective Scientific Writing Skills (15) Superior Good Adequate Inadequate None

Uses proper word choice; Voice is appropriate for audience (scientific community). (4)Writing is not personal. (The experiment is the focus/subject of the writing; not the class, individual, lab group or teacher); Writing is in 3rd person. (3) No personal pronouns used.Objective vocabulary is used at all times. Does not use subjective terminology, such as “pretty,” but uses terminology that is quantifiable and unbiased. (4)Clear sentence structure. Errors are minimal or absent and do not obstruct the meaning or intentions behind the statements. (4)Total Points for Conventions of English: _____/15

Preliminary Scientific Paper Score _____/100

Section 8: Extra Credit Magazine Cover (+9) Superior Good Adequate Inadequate None

Professional Design and Artistry

Uses High Quality Graphics in Color

Magazine Title, Date of Issue, and Volume Number

Features or references the scientific journal article and page number found

Unique features of magazines evident (example barcode, price, etc.)

Total Points Added for Extra Credit _____/+ 9

Section 9: Accuracy in Identification of Mystery Culture (-9) Superior Good Adequate Inadequate None

All cultures must be identified correctly to maintain score given for scientific paper. Negative points will be given for improperly identifying cultures as follows:1 misidentified culture = -3 points2 misidentified culture = -6 pointsAll misidentified = - 9 points

Total Points Deducted for Misidentification of Cultures -_____/ - 9

Final Total for Scientific Paper ______/100


Microbiology Notebook Grade Sheet

Notebook Criteria

0-3 4-6 7-8 9-10 Points Earned

Form

atti

ng

Neatness(no erasures, pen, etc.)

Erasures and/or data concealed; AND Pencil or non-black/blue ink used throughout. Doodling and extraneous marks.

More than two erasures or data concealment; OR pencil and non-black/blue ink used more than once. Some doodling and extraneous marks.

One erasure or concealment of data; OR one use of pencil or non-black/blue ink. No case of doodling and extraneous marks.

No erasures, all errors dealt with properly; All labs in black or blue ink. No case of doodling and/or extraneous marks.

Witnessing and Accountability for Legitimate Data

No signatures or witnessing present; Two or more pages torn out. Blank spaces not crossed out or “NWUI” noted with dates and signatures.

Missing two or more witness signatures. And/or two or more blank or unused space improperly accounted for. One page torn out.

Missing one witness signature; And/or one blank or unused space improperly accounted for. No pages torn out.

All witnessed entries signed. No pages torn out. All blank or unused spaces crossed out or “NWUI” noted with dates and signatures. No pages torn out.

Format(dated, signed, paginated, from or go-to statements)

No dates, signature, or page numberings; OR missing a significant number of dates, signatures, or page numberings. Missing from or go-to statements to tie sections of continuous lab work together.

Missing two or more dates, signatures, page numberings, or from or go-to statements to tie sections of continuous lab work together.

Missing one date or signature, page numberings or from or go-to statements to tie sections of continuous lab work together.

All pages dated and signed on every page. All pages numbered. All pages dated and signed at the end of every experiment. No missing “from” or “go to” statements to tie sections of continuous lab work together.

Table of Contents

No Table of Contents; OR Table of Contents is significantly incomplete.

Missing two or more labs in the table of contents.

Missing one lab in the table of contents.

All labs listed in the table of contents, with page numberings.

Lab Write Up

0-3 4-6 7-8 9-10 Points Earned

Labs

Objective No objective or purpose stated.

Objective and/or purpose unclearly stated. Meaning of statement obscured.

Objective and/or purpose stated. Errors do not obstruct meaning of statement.

Objective and/or purpose clearly stated.

Procedures Labs cannot be performed as drawn in the flowchart; many aspects missing; flowcharts are too small, confusing, or are difficult to discern. Drawings are sloppy. No text to supplement flowchart.

Some labs cannot be performed as drawn in the flowchart. Some aspects missing. Flow chart is difficult to discern. Drawings are sloppy. Some text supplement flowchart.

All labs can be performed as drawn in the flowchart and supplemented by text, but lacking some clarity; Drawings are good, but needs some improvements and revisions.

Labs can be performed and followed as drawn and annotated by text. Drawings are excellent, clear, without ambiguity.

(weightx 2)

Results/Data

Data not recorded or indecipherable. No relevant images.

Data recorded inconsistently. Missing clarity. Some images are included, but lacking

Data recorded, but missing no more than one or two data points. Images included and

Data recorded completely.

(weightx 2)

Laboratory Notebook Record Keeping Proceduresnewburyparkhighschool.net › malone ›...

Documents

Transcript of Laboratory Notebook Record Keeping Proceduresnewburyparkhighschool.net › malone ›...