a Bio 310WI section
Prof.
Revised F2022/F16//s17/ f19/f20/f21
General Lab Report Instructions &
Instructions for Reports 1-9
Instructions for Writing a Microbiology Report
The report has several sections and the written report should be proof-read for grammatical and spelling errors. It is much preferred that you type up the majority of the report. Keep in mind, if the report is not neatly presented i.e. if I can not read your handwriting, understand what you are trying to say, interpret your drawings and/ or descriptions, etc., I may not be able to grade the report effectively. This is obviously to your disadvantage! Lab reports are generally due 1 to 1 ½ week(s) after the lab exercise or set of exercises is/are concluded. Several lab reports include information relating to more than 1 exercise (maximum 2). In those cases (refer to the following pages), you are responsible for submitting all of the pertinent information.
Each section of your lab report should be labeled.
The sections and roles of each section are as follows:
1) Title of Report: The title should be approximately 10 words (or can be less) and should encapsulate the main focus of the lab exercise. It is not an introductory statement – it should not be in the form of a complete sentence or a paragraph! Does not have to be a separate page.
2) Introduction: You should present the scientific purpose(s) and / or objective(s) of the experiment or exercise being performed. It should also provide the reader with some background information about the subject. In some cases, you may want to provide some background information about the organism(s) being worked with and / or some theory behind the techniques being used. This section provides “context” for the exercise.
3) Materials: You should provide a short list of what you consider to be the most important items used for the exercise. This would include the specific microorganisms used for the experiment, properly written out i.e. genus and species names. Also, if any of the materials are different than those cited in the lab manual, these should be included here. This does not need to be an exact replica of the materials list in the lab manual.
4) Methods: You should describe preferablyin your own words what it is that you did in the lab. As above, if the procedure(s) differed from what was cited in the lab manual then you should report those differences here. Again, this should not an exact replica of the procedure in the lab manual. If you report that we did something that we did not perform, it will be noticed and might very well affect your grade for the report.
5) Results: You should present the outcomes of your experiments and observations without discussing their meaning or “implications”. Results can be presented as descriptions of observations, drawings with labels (a brief description is strongly recommended especially for those of you who do not draw well), tables and / or graphs. Tables and graphs should be properly labeled to understand what is being presented.Photographs alone are NOT acceptable results (this is the case regardless of whether they are your own or from Google.)
6) Conclusion/Discussion: Do NOT represent your results in this section; however, you will have to refer back to them as you “interpret” and discuss the significance of your results. For example, in performing the particular experiment or exercise, did you achieve your objective(s)? If not, why not? Can you provide suggestions for improvement? Even when the experiment /exercise did work, it is often appropriate to discuss reasons why the experiment might not work on other occasions. In most cases, it would be appropriate in this section to relate your results to existing relevant theory and / or knowledge. I will assign questions from the lab manual, which you need to answer in this section, to help with this aspect of the lab report. This will be provided to you shortly – see following pages.
7) References: Minimally, this should include the lab “manual”, i.e. lab materials being provided to you; however, in many cases, you may need to cite the textbook as well. If you are using websites and other resources, these should be cited as well. For websites, the address should be provided and the date that the website was accessed.
N.B. Sample of lab report for use of light microscope to view protozoan organisms is at end of document.
LAB REPORT 1- Selected questions forGram stain (with a little Streak Plate/ Simple Stain mixed in to the Conclusion/Discussion questions)
Note: This is turned in after you have viewed your Gram stain slides.
In your Gram stain results, please remember to tell me which two of the 3 bacteria you are working with in this exercise, which is Gram positive or Gram negative, what are their bacterial shapes, how they are arranged, relative sizes….. Bacteria for this exercise are: Escherichia coli, Bacillus subtilis, and Staphylococcusepidermidis (orsometimes S. aureusinstead. For the streak plate and simple stain exercise, we were using Micrococcus luteus instead of a Staph.)
Q1.There are significant chemical (structural) differences between the cell walls of gram-positive and gram-negative bacteria that might explain differences in the rate of decolorization. What are those differences (and with respect to gram-negative cells, there are two structures that need to be mentioned with Gram negative cells.) And note: We are not referring to the Gram stain procedure itself.
Q2.How does the age of a bacterial culture affect the interpretation of a Gram stain reaction? Explain why the age of a culture affects the Gram stain reaction.
Q3.Predict the effect of the following 4 “mistakes” made when performing a Gram stain. Consider each mistake independently. Also please explain your answers as to why you get these outcomes.
a) failure to apply the iodine b) failure to apply the decolorizer
c) failure to apply the safranin d)reversal of order of crystal violet and safranin stains
Q4.a) In the streak plate technique, how are microorganisms diluted and spread out to form individual colony forming units (CFUs) aka colonies?
b)Which area of a streak plate will contain the greatest amount of growth? The least amount of growth? Explain your answer.
If you did a streak plate, did you achieve isolation using the quadrant streak? If so, in which streak (1,2,3 or 4) did it occur? If you did not, what might you do differently next time to improve your result? Or as a substitute if you were absent the day the class performed the streak plate, answer as best as you can from what you saw from my E. coli streak plate that was passed around the room for you to observe.
Q5. What kind of stain is safranin and crystal violet? What charge do these stains have and what type of molecules do they bind to with respect to charge?
Q6,Which basic stain did you use for performing the simple stain on your two bacteria? What color did they both stain? Did both of your bacteria stain the same color? Why would that be the case for a simple stain but not for the gram stain procedure?
Note: For lab exam should: I) Predict what would happen if you apply the decolorizing agent for too short a time? Too long a time? II) understand why Gram stain is a differential stain procedure and not a simple stain procedure III) understand how/why bacterial structures of Gram positive and Gram negative bacteria contribute to their differentiation by the Gram staining process (related to this: would the terms Gram positive/Gram negative apply to a eukaryotic cell such as an epithelial cell?)
Reminders: Besides knowing the various functions of the light microscope parts, you should know what the term resolution aka resolving power is about (& threshold value for light microscope) and what is the purpose of immersion oil for the 100X objective lens.Last, what kind of stain is safranin and crystal violet? Also you should know terms:parcentric, parfocal, field of view, depth of focus.Lastly, you should have some idea as to how you would handle a microscope when you are in the lab.
Some other matters to think about:
i)What is the meaning of the term pure culture? How might streak plating allow you to obtain a pure culture from a mixed culture?
ii)What is the meaning of the term colony forming unit (CFU)? Why is it applicable when talking about the growth on a streak plate? Is a CFU always an indication of a pure culture?
iii)What does aseptic technique mean in context of working in the laboratory?
iv)Why is agar added to media such as the media we used for streak plating?
v)Why are you incubating streak plates in inverted position?
vi)What reasons might lead to absence of growth on a streak plate?
vii)A wet mount of Bacillus subtilis was demonstrated in class. Was it easy to see the B. subtilis, especially when they were dilute in the suspension? Think about their size? Think about why we use stains in the lab to help us visualize microorganisms.
LAB REPORT 2- Selected questions for Acid-fast stain
Note: This is turned in after you have viewed your acid fast stain slides.
Bacteria for this exercise are: Mycobacterium smegmatis(or M. phlei) and S. aureus.
Q1. Acid fast bacteria have some significant differences as compared to non-acid fast bacteria. What is the major chemical (structural) difference among these acid fast bacteria that might explain why the need for the acid-fast stain procedure? Hint: This structural difference also affects the way the bacteria grow on media (both broth as well as the media containing agar) and the length of time they take to grow.
Q2. Why do you suppose the acid fast stain in not as widely used as the Gram stain? When is it more useful than the Gram stain? Can you name the bacterial genera that are considered to be acid fast?
Q3. (Use the lab manual to answer this) How does heating the bacterial smear during a Ziehl-Neelson stain procedure promote entry of carbol fuchsin into the acid fast cell wall?Why is the Kinyoun procedure referred to as the “cold” acid fast stain procedure? Explain what happens instead in the Kinyoun stain procedure during the application of the carbol fuchsin.
Q4. Are acid fast negative bacteria stained by carbol fuchsin? If so, explain why the acid fast stain is considered to be a differential stain? What color are non-acid fast bacteria and why?
LAB REPORT 3- Selected questions for endospore stain and capsule stain exercises as well as the catalase assay)
Note: This lab report is turned in after you have viewed the slides for both the spore stain and the capsule stain (sometimes the capsule stain is done as a demonstration but you are responsible for looking at a capsule stain slide and knowing how it is done and important to remember, we are using the nigrosin stain procedure as described for performing a negative stain in order toto stain for capsule! With that said, in the handout, another protocol is mentioned that more directly stains for capsule. We probably will briefly mention it.). Also, this lab has 2 parts which might be done on 2 separate days. When turning in the lab, make sure you include BOTH parts. It is turned in after the capsule stain part is completed. (Note: Our lab materials claim the bacterial endospore contains keratin. It is actually a protein that is keratin-like.)
Bacterium for the spore stain is: Bacillus subtilis. Bacteria for capsule/negative stain are Enterobacter aerogenes&S. aureus.
Q1.What is the function of a bacterial endospore? Hint: How easy is an endospore to destroy? Use your textbook/lecture notes to help you.
Q2. Why does this exercise call for use of an older culture of Bacillus? What does a positive result for a spore stain indicate about the organism? What does a negative result for the spore stain indicate about the microorganism?
Q3. List three specific functions of capsules. Use your textbook/lecture notes to help you. (Hint: two of these functions are often listed as functions of various virulence factors). Note: It is not enough to say that it protects bacteria from the immune system or that it is a virulence factor.
Q4. In the capsule stain process used in class (using nigrosin), are the bacterial cells heat-fixed to the slide? Why or why not?
Q5. Eosin is an acidic red stain and methylene blue is a basic blue dye. What should be the result of staining a smear of encapsulated bacteria with a mixture of eosin and methylene blue?
Please note if the capsule is stained or not stained at all.
Q6. What chemical reaction does the catalase enzyme carry out, i.e. what is the enzyme’s substrate and what are the end products? Why is this enzyme beneficial to the organism producing it?
For lab exam: you should have a general idea regarding: the bacterial spore cycle (its different stages).
As a somewhat related question to Q1, is endospore formation a form of bacterial reproduction or is it doing something else for the bacteria? Explain your answer. Also you should understandwhy an acidic stain such as nigrosin or eosin, which in many cases is being used for a negative stain procedure, does not colorize the cells in a bacterial smear.
As mentioned in lab, how does India ink work as a negative stain (as compared to nigrosin)?
NOTE:This next section will NOT be part of a lab report but info. is needed for lab exam 2.
Selected questions/ information from for a module dealing with bacterial growth and culture characteristics – including example of table for thinking about pigmentation information (see Table below)
This exercise on “culture and growth characteristics” is usually done as a demonstration and this year may end up primarily being handled in reference to handout. Some questions are provided below regarding the various cultures to help you make appropriate observations.
Some of the bacteria for these exercises are usually: Escherichia coli, Serratia marcescens, Micrococcus luteus, Bacillus subtilis, Pseudomonas fluorescens (or P. aeruginosa), Mycobacterium phlei (or M. smegmatis) and sometimes S. aureus. If M. smegmatis and/or P. aeruginosa is used, there will be added explanation. Information for Staphylococcus aureus and S. epidermidis as well as Enterococcus faecalis and Proteus vulgaris will also likely be mentioned.
Growth/culture characteristics exercise results: FILL IN information for “Pigmentation Table” below
Incubation Temperature of the cultures
Colony color produced Colony color produced
Microorganism| i.e. pigment at 25ºC |pigment at 37ºC
Serratia marcescens | |
Micrococcus luteus | | info not required
Pseudomonas fluorescens | | info not required
Mycobacterium phlei | | info not required
N.B. These next questions are not identical to the ones provided during the lab session but answering those questions during lab should help you in answering these questions (which help prepare you for lab exam, etc.)
a)In broth, does M. phlei grows in suspension, i.e. does it cause the broth to become turbid throughout the entire tube? Do the colonies on solid media look flat like other colonies? Explain your answer.
This somewhat relates to term in matching exercise below, friable.
b) Are all colony forming units (CFUs) transparent? Which are not?
c)Which CFUs have irregular edges (“wispy” edges) suggestingthat the bacteria might be motile? This relates to terms below in matching (filiform colonies vs. colonies with spreading edges).
d)Which bacteria that we observed do not produce pigment?
In discussion, include answers to:
Q1. How does complex medium compare to synthetic (chemically defined) media? What kind of medium were you using in the exercises such as the staining exercise and the streak plate/pour plate exercise?
Q2. Define or explain:
a) obligate anaerobeb) facultative anaerobe
Q3.Match the following terms from exercise 2-3 in lab manual usually in use
______ filiform 1. Rough texture with a crusty appearance
_____ _spreading edge 2. Solid growth seeming to radiate outward
______ transparent 3. Smooth texture with solid edge
______friable 4. Almost invisible or easy to see light through
______pigmented 5. Produces colored growth
Q4. Match the following terms from exercise 2-4 in lab manual usually in use
_____Flocculent a. Growth at the bottom
_____Sediment b. Growth at top around the edge
_____Ring c. Evenly cloudy throughout
_____Pellicle d. Suspended chunks or pieces
_____Uniform fine turbidity e. Membrane at the top
Note for lab exam:Should have understanding of i) besides pigment production, possible other culture characteristics that can be useful in identifying a bacterial species (photos in handouts, etc. can be very helpful here). Some other terms to know: mucoid, punctiform, filamentous.
LAB REPORT 4 part A- Fermentation (phenol red glucose broth/ oxidation-fermentation [OF]media)
Note: This exercise will be combined below with the exercise on TSIA and SIM (Lab 4 part B). The report instruction are following the lab report 5 instructions (Part B is being done after we do the lab work work for lab report 5. I will explain why in class.).
***Note: for lab report 5 – there will be additional guidance provided in lab.***
LAB REPORT 5- Selected questions for exercise dealing with three different types of selective / differential media as well as TSA
(N.B. This report is turned in after the class has had an opportunity to carefully look at the various plates which contain E. coli, Proteus vulgaris (PV), Enterobacter aerogenes (EA), Pseudomonas aeruginosa (PA), Micrococcus luteus (ML), Staphylococcus aureus (SA), Staphylococcus epidermidis (SE) and Enterococcus faecalis). Note: EC, PV and EA are considered to be enterics.
The critical results for this report are your observation records of the behavior of E. coli (EC), S. aureus (SA) and P. vulgaris (PV) on the 4 viewed media: Tryptic Soy Agar (TSA), MacConkey (Mac), Hektoen and Mannitol Salt Agar (MSA) as well as PA on Hektoen and Macand SE on MSA and TSAas well as ML on MSA and TSA.
You should be able to explain/describe: a)whether they all growing equally well on all types of media i.e. which plates are the bacteria growing on and which plates they are not b)you should know the ingredients in the different media that affect the bacterial growth (and there is a table in the lab manual that helps you on this point) c)if the bacteria are growing on the media, you should be able to describe their appearance ex. what color is the colony / media surrounding the colony. You need to explain what is happening to the media /why these changes are occurring– essentially what are the biochemical activities of the bacteria that lead to these changes. With respect to the above, it would be wise to include a little description with respect to their selective and differential ingredients for the following media: MacConkey, Hektoen and MSA.
Some questions are being provided to you to help you make the correct observations. These questions should be used for each of the microorganisms on the different media. You are responsible looking at all of them – not just the ones you inoculated. There are also additional questions at the end of this section that overlap somewhat with what I am looking for in your results (you will get “credit” if you get correct below).
1.Does the bacteria grow on this medium? (There are 8 bacteria & 5 types of medium.)
Be careful not to mistake streak marks for bacterial growth!
2.If not, why not? (i.e. what is in the medium that prevents it from growing)
3.If the bacteria grows on the particular medium (MacConkey and Hektoen),how is it growing?
i) what color is the colony and how does the color compare to the original color of the medium.
ii)if there is a color change, what does that mean in terms of the bacterial metabolism? If there is no color change, what does that mean? (Hint: With respect to MacConkey, think about the differences observed between E. coli (or E. aerogenes) in contrast to Proteus and Pseudomonas. With respect to Hektoen, why doesPseudomonas look different from E. coli and Proteus (and E. aerogenes)?
4.In the case of bacterial growth on MSA, it is more relevant to observe the change in color of the medium itself surrounding the bacteria colony.
i)Describe the colony color of both S. aureus and Micrococcus luteus. Now describe the color of the media surrounding the colonies. Is there are a difference? Why?
ii)Now describe what is happening with S. epidermidis on MSA. Does S. epidermidis look different than the other two? Can you explain?
Selected questions for conclusion / discussion section:
Q1. Explain the purposes of selective media and differential plating media.
(Think about what was said in lab about the purpose of MacConkey media ingredients, etc.).
Q2.What purpose does the nutrient agar plate serve with respect to the various selective / differential agar (MSA, Hektoen and Mac)? How about the benefits of using blood agar?
Q3. What percentage of NaCl is there in MSA? What would be the likely consequence of omitting the NaCl in MSA? Why? Would all organisms grow on MSA if there was no or very little NaCl?
Q4.Proteus species and certain species of Salmonella produce a black precipitate when growing on Hektoen. Chemically what is occurring to cause the black precipitate to form? What ingredients in the media are being used by the bacteria to produce the black precipitate?
Q5. With respect to MacConkey agar, what would be the possible consequence of:
a)replacing the lactose with glucose? Would you be able to distinguish EC from PV if there is glucose instead? Explain. (Exercise 7 results will help here with this question).
b)replacing the neutral red with phenol red (yellow when acidic; red or pink when alkaline)?
Additional questions that some students feel useful to answer (helps with providing the information for the results for this exercise):
6. Which microorganism that you observed in class produces hydrogen sulfide (or at least theoretically)? How would this trait be observed with the media we used?
(also useful to think about: which amino acid can provide sulfur to bacteria?)
7. a)What is the meaning of the color change to the media and/or colony when E. coli is grown on MacConkey agar? Or Hektoen? How about Pseudomonas aeruginosa on MacConkey or Hektoen? How about Proteus on MacConkey or Hektoen?
b)How about Staph. aureus on Mannitol Salt Agar (MSA) in comparison to Staph. epidermidis or Micrococcus luteus on MSA (in this case you have to look at image e-mailed to you)?
8.Did you see any swarming of Proteus on blood agar or other media?
Note: For lab exam, it would be useful for you to understand:
i)what are the ingredients in MSA, Hektoen and MacConkey supplying carbon and/or nitrogen to the bacteria growing on the media iii) iv)knowing which ingredients in MSA, MacConkey and Hektoen agar make the media selective or make the media differential and knowing which bacteria grow or don’t grow on these plates and if they grow, have some knowledge as to how they would look ( you will get some guidance here in lab)
LAB REPORT 4 parts A and B- Selected questions from exercises on two different types of fermentation media & multiple-test media).IMViC reactions will also be covered in this exercise, at least briefly.
Bacteria to be used in exercises regarding the fermentation media, phenol red glucose media and the OF media,include: E. coli, Pseudomonas aeruginosa, Proteus vulgaris
Bacteria to be used in the exercises regarding the SIM and TSIA media include: E. coli, Enterobacter aerogenes, S. aureus, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens (the specific bacteria depend on the media.)
Q1.All enterics are facultative anaerobes. What color results would you expect for organisms in O-F glucose media inoculated with an enteric. Remember to describe both sealed and unsealed tubes. How would an obligate aerobe appear in both the sealed and unsealed tubes? What would happen if lactose were used instead of glucose? Would you expect the results for E. coli and P. vulgaris to be identical? Explain based on your knowledge from how these two bacteria grow on MacConkey media.
Q2. How might the results obtained in exercises such as this one or the exercise on extracellular enzymes be used for the identification and/or classification of bacteria?
Q3. a)With respect to the phenol red glucose media, what is the Durham tube and what is its purpose?
b)What is the purpose of the mineral oil for the OF media?
Q4.List two advantages of a multi-test system such as triple sugar iron agar or SIM.
N.B. To help you figure out the results of these two exercises, you should fill out the following tables for your lab report using the information that you learned in lab.These tables are not the same format as in class. I want to see that you really understand the material.
Results: 2 Tables relating to the OF media. Remember: the OF assay is done with pairs of tubes so a microorganism is introduced into 2 OF tubes and one tube receives an overlay of mineral oil!!!)
Table for 4-part A -1. Which microorganism(s) give the following results for this pair of tubes?
Is this bacterium oxidative Which of the used bacteria
Medium + or – mineral oil Reaction or fermentative? show this reaction?
OF tube /without mineral oil Yellow
OF tube/with mineral oil Green
Table for 4-part A -2. Which microorganism(s) give the following results for this pair of tubes?
Is this bacterium oxidative Which of the used bacteria
Medium + or – mineral oil Reaction or fermentative? show this reaction?
OF tube/without mineral oil Yellow
OF tube/with mineral oil Yellow
Results: 3Tables relating to the phenol red glucose media
Interpretation
Is sugar fermented?Bacteria used in class with
Medium Reaction i.e. acid/gas/neither? this reaction if any
Phenol red /w glucose Yellow – no air bubble
(or very small bubble)
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
Phenol red/w glucose Yellow – with air bubble
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
Phenol red/w glucose Orange – no air bubble
Table relating to SIM media (adapted from Table 26-6 on page 235 in Wistreich manual)
Which of the used bacteria
Medium Reaction Interpretation show this reaction?
Sulfide indole motility a)Blackening of the medium
(SIM)
– – – – – – – – – – – – – – – – – – – – – – – – – –
Medium stays yellow
__________________________________________________________________________
b)Red color formed with addition of
Kovacs reagent
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
The layer containing the Kovacs
Reagent stayed yellowish
___________________________________________________________________________
c)Growth only on line of inoculation
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
Growth throughout the tube
Table relating to TSIA media (Triple Sugar Iron Agar)
Note: This table is also adapted from Table appearing in Ex. 26 of Wistreich manual)
Which bacteria (if any)
Medium Reaction Interpretation show this reaction?
Triple Sugar a) Yellow butt, slant red
Iron Agar – – – – – – – – – – – – – – – – – – – – – — – – – – – –
(TSIA) b)Yellow butt, yellow slant
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
c) large holes in the agar
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
d)formation of black precipitate
– – – – – – – – – – – – – – – – – – – – – – – – – – – –
e)Red butt, red slant
Note: For exam i)would be a good idea to understand difference terms, oxidative organism vs. fermentative organism ii)know name of the pH indicator used in the media used in Exercise 5-2 and where you saw it in other media iii)how might the results obtained in these exercises be used for the identification and/or classification of bacteria.
LAB REPORT 6- Selected questions for media testing for production of certain extracellular enzymes. As a side note, the previous exercises were more concerned with production of intracellular enzymes.
(In your results, do not just say positive or negative reaction for the various different bacteria on the 4 different media. Explain the color / media changes that are occurringand explain what is happening biochemically. You are responsible for knowing about the theory behind all 4 media as well as the results of all 4, unless told otherwise.)For the bacteria growing on blood agar, please note what their hemolysis pattern was and why.
Bacteria being used in these exercises will include: Bacillus subtilis, E. coli, S. aureus, S. epidermidis, (and Serratia marcescens will likely get a mention). In class you will get further instructions.
Q1. Bacteria can produce extracellular enzymes. What role do these extracellular enzymes play in bacterial metabolism?
Q2.Suppose you had poured iodine on your plate and noticed clearings in the uninoculated area, as well as around both of your transferred cultures? What are some possible explanations for this occurrence? Was the integrity of the exercise compromised? What kinds of things might be done to avoid this problem in the future?
Q3.Is it acceptable to read a positive test before the incubation time is completed for an assay such as the milk plate assay? Why is this not the case in a starch agar assay? How about an early negative result?
Q4.Why is it advisable to use a positive control along with organisms that you are testing
Q5. With the toluidine blue agar mentioned in class, what reaction is being tested for here? What kind of dye is toluidine blue? If the medium turns from blue to pink, what does that mean biochemically?
Q6. For these questions refer to introductory pages of Ex. 5-20:
a)What happens to blood agar when an alpha-hemolytic reaction occurs?
b)What happens when a beta-hemolytic reaction occurs?
c)What happens when a gamma-hemolytic reaction occurs?
Note: For lab exam: i) what does the enzyme casease (a type of protease) have in common with amylase ii)how do you know these are exoenzymes rather than working in the cytoplasm iii) besides the term depolymerization (disassembly of DNA), what other term can be used to describe this process
iv)With respect to the Starch Agar assay, how would you expect the results to change if you were to add glucose to the medium (in addition to the starch already present)?V) How might the results obtained in these exercises be used for the identification and/or classification of bacteria?
NOTE: This next section regarding the Phage/serial dilutions/plaque assay (based on handouts)
will not be part of a lab report but some of this info. is useful for lab exam 4.
Some questions/terms to think about:
1.What is meant by the term plaque-forming unit (PFU)? What is the difference between PFU and plaque?
2.How many PFUs per ml are there in a sample that was diluted 10-8 and has given 100 PFUs when 0.1 ml of the 10-8 diluted sample?
3.What is the purpose of incubating the bacteria and phage suspension in the cold in the presence of calcium salts?
4.Why are the bacteria and diluted phage mixed into soft agar and then plated out onto a media surface containing a higher amount of agar?
5.Did you see any plaques on the negative control plate? Please explain?
6.As you made successive dilutions and plated the phage/bacterial suspensions, what happens to the number of PFUs from lowest dilutions to highest dilutions?
7. Differentiate between the terms: lytic cycle and lysogenic cycle. What can happen if a lysogenized bacterium is stressed?
8.What is the difference between a virulent phage vs. temperate phage? What is a prophage?
9. Why do we talk about virus replication in terms of a one-step growth curve?
10.What are the steps of the lytic cycle? Lysogenic cycle?
11. How does the lytic cycle relate to generalized transduction and how does the lysogenic cycle relate to specialized transduction?
LAB REPORT 7- Selected questions for exercise on how we test for antibiotic resistance
( antibiogram, etc.)
Note: Results for this exercise would include explanations of the Kirby-Bauer and E test i.e. what is the purpose, how are they set up. What kinds of results did you observe for each type of assay?
Drawings can be of help. A table containing Kirby-Bauer plate data will be provided at the time of the exercise to make it possible for you to interpret the data. Using the provided data, you should be able to say who is resistant, sensitive or has intermediate resistance. Further explanation will be provided later.
NOTE: Arrows on chart on next page might not apply this semester (i.e. you may be asked to substitute Streptomycin for ampicillin.)
Example of how the table would be set up. * Note, there may be different antibiotics mentioned in class & materials..(Note: mcg = micrograms aka µg)
S. aureus (diameter in mm) E. coli (diameter in mm)
*S 10 mcg/ml
*E 15 mcg/ml
*P 10 mcg/ml
*Te 30 mcg/ml
*C 30 mcg/ml
Q1.Are antibiotics considered to be effective against virus infections? Explain why or why not.
Q2. What is the benefit of using Mueller-Hinton medium in a Kirby-Bauer assay instead of media such as nutrient agar or tryptic soy agar?
Q3. What is the function of the enzyme beta lactamase with respect to a beta lactam antibiotic? Where are beta lactamases found in a bacterial cell – are they typically intracellular or extracellular (secreted) enzymes?
Q4.a)What does the abbreviation MIC stand for? Provide a brief explanation of its relevance to bacterial drug resistance.
b)Explain what an E-test is. How is it set up and how can it be used to determine a MIC for a bacterial organism.?
c)Using the text,you will see there is at least one other test that can be used to determine a MIC for a bacterial organism. Please give a brief description of that assay.
Q5.The antibiotics referred to above as “S”, “E”, “Te” and “C” all have the same cell target (note, S, E, Te and C are usually included in this exercise but sometimes one or more have substitutes). What part of the cell do the S, E, Te and C antibiotics bind to? What process do they inhibit? If other antibiotics are mentioned in the class data, what are their cell targets?
Note: For exam, it may be of use to know: i) what is a monobactam & why/when is it used?
ii) what is clavulanic acid & how/ why is clavulanic acid used iii) how can one relatively easily test for the ability of a bacterial cell to produce a beta lactamase? (In this case, you may have to look up this answer by Googling, etc. – sometimes we get to do a demonstration so be on the lookout….) iv)Typically, what kind of genetic element carries the gene for beta lactamases – is it on the chromosome, on a plasmid, etc.? Can it be associated with transposons? v)think about how this relates to R factors and superbugs.Vi) Excluding the situation with respect to allergies, is penicillin usually considered to be nontoxic to humans? Explain your answer.Vii)
This lab report 8 may be used as an extra credit lab report (more about that in class) or as a substitute for one of the other required lab reports.
LAB REPORT 8- Selected questions from exercise on algae, protozoa & fungi&rotifera
(N.B. Minimally, your results are drawings of: at least one of the fungi shown on the commercially prepared slidessuch as Aspergillus niger, Penicillium notatum, Rhizopus nigricans in its asexual form and Rhizopus nigricans in its sexual form; Saccharomyces cerevisiae (if available) i.e. not culture plates and at least one representative protozoan and one algae from pond water (or from pure cultures if that is what we have available) and / or from commercially prepared slides of some protozoa such as paramecium and Plasmodium falciparum and algae such as spirogyra or volvox. We will have some other microbes, etc. available for these exercises and also the multi-cellular organism, rotifera.
Note: Lecture one material may be helpful here to answer some of the questions below.
Q1.Some of these eukaryotic microorganisms have cell walls and one does not? Which one does not and what does it have instead? If it has a cell wall, what is its biochemical composition?
a) protozoab) algae c) fungi [ d)bacteria ]
(Bacteria are here for contrast.)
Q2. Besides the presence or absence of cell walls (or varying composition of cell well when present), how are fungi different from both algae and protozoa? In other words, what are some distinguishing characteristics of fungi that make them different from algae? Fungi different from protozoa? Algae different than protozoa? Some of the characteristics that can be relevant: i)photosynthetic vs. non-photosynthetic ii)unicellular / multi-cellular iii) asexual vs. sexual reproduction
Q3. Give the function or functions of the following:
a) chloroplast b) cilium found on/in protozoa
Q4: What are the 4 different ways that protozoa move? Hint: The 4th way pertains to Trypanosomes so look at the figure in the handout and some of the information pertaining to them.
Q5. What type of cell organization do multi-cellular fungi exhibit –is there cell differentiation? There are different types of filaments that have different functions. Please explain the difference in function between an aerial (reproductive) hypha and a vegetative hypha. Distinguish between a hypha(e) and a mycelium.
Note: For exam: i) be able to distinguish the function of these two types of vacuoles a)contractile vacuoleb)food vacuoleii) be able to name two forms of asexual reproduction associated with yeasts iii)know what is a pseudomycelium (also known as a pseudohyphae) iv)know how do the words conidia and sporangia relate to fungi v)know what the word conjugation mean when we are referring to protozoa such as paramecia vi) know which yeast produces red colonies (may need to look at handout)
Sample Lab Report
Lab Report #: 0
Title: Using the microscope to view protozoan organisms.
Introduction: Most microorganisms are very small microorganisms and invisible to the naked eye. Use of the microscope allows us to magnify these microorganisms by 4-fold to 1000-fold. With protozoan organisms that are appropriately stained, use of the microscope allows us to see shapes of the microorganisms as well as some of their internal organelles. Among the purposes of this exercise is to re-familiarize us with the use of the microscope as well as reacquaint us with basic details about these protozoan microorganisms.
Materials: Prepared slides of amoeba, paramecia and euglena. Other equipment included: compound microscope, lens paper.
Methods: Commercially prepared slides of amoeba, paramecia and euglena were viewed under the microscope under 4X, 10X and 40X objectives.
Results***:
Drawing of amoeba Drawing of paramecium Drawing of euglena
***Note: I used to draw these in but if I wanted to send the file electronically the information was lost. Therefore, here, I am substituting these drawings in for the real thing but you should not do that for your lab reports.
Note: The paramecium features are not labelled but some visible features are its cilia, the contractile vacuole, a food vacuole, its gullet and nucleus. For the euglena, it is hard to see the labels unless you enlarge the figure. Some of its labeled features are: the flagellum, its nucleus, its contractile vacuole, its eye spot and several chloroplasts..
Discussion: As the magnification of the lenses increased, it became easier to discern the different characteristic shapes of these three different protozoans and some of the details associated with the protozoan. These protozoans were stained so it was possible also to see sometimes organelles. With the amoeba, it was possible to see the pseudopodia and the darkly stained nuclei. With the paramecium slides, it was possible to see their characteristic slipper shape and with some slides – nuclei and cilia. With some paramecium slides, it was possible to see the paramecia carrying out sexual reproduction i.e. conjugation. With the euglena (which sometimes are stained red [in nature, they would be green]), it was possible to see their characteristic thin, tapered shape (and depending on the staining, possibly even the eye spots).
GENERAL LABORATORY CONDUCT FOR MICROBIOLOGISTS
The objectives of the special procedures employed in microbiology laboratories are:
(1) to prevent introduction of undesirable organisms into the supplies and cultures being used (that is, to prevent contamination);
(2) to prevent the possibility of infecting people working in the laboratory, including those who do the cleaning-up, by the organism cultured in that laboratory. Even though the organisms studied in an introductory course are not pathogenic (or only weakly so), it is prudent to treat them as you would a pathogen and not to develop careless habits.
The following guidelines will help you meet the above objectives.
1. Always read the assigned laboratory material before the start of the laboratory period.
2. Come to the laboratory properly dressed – never with bare feet, loose long hair or loose clothing.
3. Do not eat, drink or smoke in the laboratory, and do not place pencils or similar objects in your mouth. All students are required to wear lab coats, safety glasses and proper footwear.
4. Do not place unnecessary items such as books or clothing on the bench tops.
5. Clean the bench tops with disinfectant before and after your laboratory work, and wash your hands before leaving the laboratory when finished.
6. Discard all cultures and used glassware into the container labeled CONTAMINATED. (This container will later be sterilized.) Plastic or other disposable items should be discarded separately from glassware, in containers to be sterilized.
– Never place contaminated pipettes on the bench tops.
– Never discard contaminated cultures, glassware, pipettes, tubes, or slides in the wastepaper basket or garbage can.
– Never discard contaminated liquids, or liquid cultures, in the sink.
7. If you are in doubt as to correct procedure, double-check the manual. If doubt continues, consult your instructor. Avoid asking your neighbor for procedural help.
8. If you should spill or drop a culture, call the instructor immediately (do this, should any type of accident occur).
9. Report any injury to your hands (before you begin the laboratory work or during the session, depending on when it happened).
10. Students are not permitted to remove specimens, cultures, or equipment from the laboratory, under any circumstances.
11. Before leaving the laboratory, carefully wash and disinfect your hands.
Two other behavioral characteristics which will enhance not only your own enjoyment of the course, but also that of your fellow students and instructor are: coming to class on time; and being prepared.
Personal conduct in a microbiology laboratory should always be quiet and orderly. Thoughtful attention to the principles of safety is required throughout any laboratory course in microbiology.
RULES ON THE USE AND CARE OF THE MICROSCOPES
A) Microscopic objectives are never placed in dyes or stains such as Methylene blue or blood.
B) The oculars, objectives and the condenser are never cleaned with Bibulous paper, Kimwipes, or harsh detergents. Only use the lens tissue which has been provided.
MICROSCOPIC CARE AFTER USE:
1) Turn off the light.
2) Remove the slide from the stage.
3) Return the scanning objective (5x) over the aperture.
4) Rotate the bottom knob of the mechanical stage to make sure the slide holder mechanism is not jutting out.
5) Place your hand on the plug and disconnect the power source. NEVER PULL ON THE CORD TO DISCONNECT.
6) Wrap the cord around the microscope.
7) All microscopes are numbered, check your number.
8) Using one hand to grasp the arm of the microscope, and the other hand under the BASE for support, RETURN YOUR SCOPE TO ITS PROPER NUMERICAL COMPARTMENT.