lab report microbiology

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a4BM005 – Microbes and immunity – Course Work assessment instructions

 

Laboratory Practical Report.

 

When communicating results findings or new techniques scientists use the written format of the journal article. These come in a variety of different styles depending on the actual print publications requirements in which the article appears. However, every scientist began and practiced their scientific writing through the most basic form of journal article style writing called a laboratory report. This details the work carried out from a series of experiments or laboratory sessions and are a favoured way for undergraduate scientists to communicate the results of their work in course work assessments.

A laboratory report has a formal structure that consists of a descriptive title, an abstract summarising the work, an introduction containing background that provides information to aid in the understanding of the rest of the report including the aims and objectives of the work and a materials and methods section giving details as to how the experiments were carried out in detail. It also includes a results section showing the findings from the methods employed and a discussion and conclusions section detailing the implications or meaning of the results gathered along with the final implications of the findings. Finally, the report finishes with a references section giving the published sources that you have used in production of the report. A more detailed description of each of these sections is given below in relation to the requirements of your course work assessment.

Your course work task is to produce a formal laboratory report based on the final laboratory practical session on your module which is an introduction to microbial identification

The Laboratory report must include:

  • A descriptive title along with your name and student number. Be sure to give a tile that says what you have done not just ‘lab report’ or ‘experiment 5’ or ‘practical.’
  • A very brief abstract or summary of the work. Keep this very short. It should contain an introductory sentence a mention of the methods used, key results and finally the main conclusions of the work.
  • An introduction. This provides back ground to the experiment you perform. This section also sets the scene for the reader and should provide enough detail to allow the reader to understand the work which is to come. As you will be using information sources from the literature you must provide in text references or citations as to where the information you use is from. This must be in Harvard format. Please ensure this is your own work and remember copy and pasting from somebody else’s work into your own is plagiarism. Remember, if you use diagrams or figures in this section the source must be correctly cited. Please refer to the referencing guide found on the WOLF page for how to use the Harvard system correctly.
  • A materials and methods section. This must be written as a paragraph not a bulleted list of instructions or consumables. Although you have been provided the methods you have used in the Laboratory work book these must be rewritten in your own words. It must also be written in the third person passive tense. This means that you never write ‘i carried out the experiment’ or ‘we carried out and experiment’ you say ‘the experiment was carried out or ‘experiments were performed to investigate’ etc. There must be sufficient detail in this section for the reader to use the work to repeat the experiment in its entirety.
  • A results section. This must only contain the results of your work and not discuss in any way why the results are the way they are. It must contain an explanatory narrative that guides the reader through the presentation of the results. Results should be presented as tables and figures that are numbered and have an explanatory title underneath. This is called a legend and should provide enough information for the reader to understand the content in the figure or table. Figures and tabulated data must be clear legible and provide accurate data from your experiment.
  • A discussion and final conclusions section. This section allows you to explain why the results are the way they are and provides a place to explore any problems or limitation with the work carried out. Here, you must discuss your work in a critical way and simply not provide more introduction or another description of the results. Your points for discussion must be supported by appropriate citations. For example if as part of the practical session you gathered results that showed the different reactions of a bacterial organism to growth in solutions containing different sugars then in this section you need to explain why there is this difference. Here you should also give the final conclusions of the work that give the answer to the problem set which in this case will be the identity of a microorganism that you have determined through the methods you have used.
  • A References section. This must include details of all the cited work used in production of the report. Although of course we are in the modern age of internet based resources like web pages its preferable here to use published work such as books and especially journal articles to support your report. Your lectures, the practical work book or non-peer reviewed information such as Wikipedia or patient information style web pages are not to be used. The Harvard system of referencing must be strictly adhered to at all times.
  • The report must be no longer than 1000 words +/- 10%. This does not include your references section.
  • It must be word processed in Microsoft word format and presented in 12pt Times New Roman font and be double spaced.

It is recommended that for convenience and to aid in production of your report you follow the structure and headings provided.

You must hand in this report electronically to your module WOLF page before midnight of the date stated on the Module time table and E-vision.

 

Suggested structure and tips for your Laboratory report

 

Please provide the information required and use the section headings shown.

To help in production of the report and understanding of the journal like format that it follows it is recommended you visit the Bioscience Horizons journal web site and browse through the freely available journal articles you will find. These have all been produced by undergraduate students and as such are written in a simpler format. By reading a selection of these you will see the kind of language used and get an appreciation of the use of the third person passive tense particularly in the methods section.

Your work must have the following structure and content;

 

Name and student number

 

Title of the report

Remember to keep this descriptive but short.

 

Abstract

Remember this is a very short summary of your work including some introduction methods, some results and the final conclusion. No citations should be used in an abstract and the abstract does not have a section number.

 

  1. Introduction

Notice this section is the first numbered section the abstract does not have a section number and stands alone.

Remember, the introduction provides the story to your work. It must be factual well ordered and well written in good English. It should provide enough information to understand why you are carrying out the work in the first place. It should also of course be supported by citations and references. This section should finish with the aims and objectives of the experiments you have carried out.

 

  1. Materials and methods

Remember this section should give enough information for someone to carry out your experiments exactly and be written in the third person passive voice as a well formulated set of paragraphs and not a bulleted list.

  1. Results

Remember here to just say what you see never try to explain why. Provide explanatory narrative to guide the reader through the results not just results tables or figures. You will see how this is done by looking at the articles from the Bioscience Horizons journal. A suggested format for the results of the experiments is a simple table summarising the data you have collected to identify your bacterial pathogen. Don’t forget to include numbered legends explaining what the figure or table is or contains.

 

  1. Discussion and conclusion

Remember, this is where you can restate the aims of the work to remind the reader what your talking about and begin to explain why the results are the way they are using published work from books or journal articles to back up your claims. Note: do not just provide more introductory content or just restate the results you must discuss them critically.

 

  1. References

This section seems to be one of the hardest to get right at first so please follow the supplied referencing guide very carefully. The Harvard referencing format must be used at all times.

 

 

 

 

 

 

 

 

 

 

 

 

 

Indicative marking criteria for the Laboratory Report Course work assignment used by the staff to mark your work.

Section and weighting/score distribution  

Abstract

(10%)

 

Introduction

(20%)

Materials and Methods

(20%)

Results

(20%)

Discussion and conclusions

(20%)

Referencing

(10%)

Excellent

 

(70 – 100%)

Extremely well written and concise abstract containing all appropriate content providing a total précis of the work including the aims introduction methods results and conclusions. Excellently written introduction in clear well structured sentences that provides background adding to the understanding of the work using suitable sources Extremely well written with all detail provided as to accurately repeat the experiments performed all in correct tense throughout. Excellent results section containing a well written narrative and suitable tables numbered table legends and correct units Extremely well written discussion and conclusions section that critically explores the results gained in the experiments performed using creative interpretation of printed literature. Excellent use of referencing through out including the use of primary journal articles in introduction and discussion.
Very Good

 

(60 – 69%)

Very well written abstract that contains only minor faults. All appropriate content presented. Very well written section with minor faults in presentation supported by good sources.

 

Essentially complete and well written methods section with an occasional minor fault. Very good results section containing narrative and tabulated data with only the occasional fault in presentation Very well written critical discussion and conclusions showing good insight and understanding supported by references. Harvard system used in text and in list with very minor faults.

Primary sources used.

Good

 

(50 – 59%)

Well-structured abstract with some content omitted usually present in the abstract. Introduction is well written in most places but does have structural issues in others such as repetitions. Good methods with some minor faults in tense usage or occasional item making the method difficult to follow Good results section with a good attempt at the inclusion of a narrative and appropriately tabulated data with some errors Good discussion and conclusions with an attempt at critical discussion of the results supported by more than one published source. Harvard system used throughout but with some minor inconsistency. Some use of web resources.
Average/ Pass

 

(40 – 49%)

Some suggestion of aims of experiment with important results and conclusion only Some significant attempt  at producing the introduction but contains issues in English usage and structure. Few less appropriate sources used. An adequate methods section with some faults in tense usage and an item missing that may preclude repetition of the experiment. A brief narrative sentence included with tabulated results only. Table legend present but insufficient in description of content A fairly superficial attempt at critical discussion with at least 1 supporting reference. Overly concise showing a lack of insight In text citations and reference list presented but inconsistent use or the Harvard system in text and in list. At least one primary resource used.
Poor/ Fail

 

(30 – 39%)

Abstract only provides introductory content and omits all other required information Some  obvious attempt at providing an introduction but badly structured and in poor English with no use of published sources Some attempt at providing a methods section but very poor and not presented in the third person. Significant faults that would preclude repeat. Some attempt at presenting results but inadequately tabulated and no narrative text. Or table legend. Some attempt at presenting discussion points but not critical or substantiated by the use of supporting sources. Contains mostly introduction No in text citations used and references in list not in Harvard format. Extensive use of only web based resources.
Very poor/ Fail

 

(0 – 29%)

No abstract presented or so poorly written it adds nothing to the report. Poorly written overly short section in poor English containing inadequate background information  and no literature sources. No methods presented or contain fundamental errors precluding any repetition of the experiments undertaken. Results section either omitted or very poorly presented such as not tabulated and no narrative present. Overly short section with no discussion points or just badly formatted introductory information. No references used in text or as a list.

 

 

Your name and student Number:

 

 

 

Session 3: Quantification and identification of bacterial organisms

 

Please note this session involves the use of small quantities of Beta-lactam antibiotics. If you have an allergy to these or any other antibiotic please inform a member of staff before you begin.

 

All experiments in this session will be carried out in pairs

 

3.1. Counting methods for bacterial cells

 

Although we can isolate microorganisms from the environment in liquid and on solid media in many cases, it is useful to be able to actually count the amount of live or viable bacterial organisms in the sample. There are many methods of assessing samples for viable bacteria. This exercise aims to introduce you to three of the main viable counting methods most commonly used.

 

 

3.2. Serial dilution of liquid bacterial cultures

 

When bacterial cells divide in a liquid growth media growing under ideal conditions of temperature and nutrition a very high number of cells per millilitre can be achieved. This of course makes counting all of those organisms in that millilitre impossibly time consuming. So to allow us to do this in a more sensible way we must dilute the sample until a concentration is reached in which we have a number we can count. We do this be a process called serial dilution.

 

A series of 10-fold (or decimal) dilutions of the culture are prepared in an isotonic salt solution, this is achieved by adding 1 part of the original culture to 9 parts of the diluent. It may be 1 ml of culture and 9 ml of diluent or 0.5 ml of culture and 4.5 ml of diluent. The resulting dilution is 1/10 the concentration of the ‘original’ and is usually referred to as a 101 dilution – 10-1 = 0.1. This 10-1 dilution is then diluted again by a factor of 10 so the resulting suspension is 1/100 the concentration of the ‘original’ or a 10-2 dilution. This process is repeated as many times as is necessary to produce the dilution series.

 

 

 

3.2.1. Finn Pipettes

 

 

To dilute your bacterial cells you will need to use a piece of equipment called a Finn pipette. It will be essential to become familiar with the correct operation of the Finn pipette as you will be required to use these in the following practical exercise. Please ensure you watch the video demonstrations provided.

 

 

 

Note: To see a video of the use of a Finn pipette click on the link below

 How to use a Finn pipette

 

 

3.2.2. Serial dilution method

 

  1. Take 4 dilution blanks containing 4.5ml of sterile isotonic buffer and label them in series 10-1to 10-4.
  2. Take your sample of coli culture and mix by swirling gently to give a homogeneous suspension.
  3. Using a Finn pipette, add 0.5 ml (500µl) of the coli to the tube labelled 10-1and mix by pipetting up and down briefly. Discard the tip to a biobin. Thus a 1 in 10 or 10-1 dilution has been prepared from the original sample.
  4. Take a fresh sterile Finn tip and withdraw 0.5 ml from the 10-1dilution and transfer to the tube labelled 10-2 and mix by pipetting up and down briefly. Discard the tip to the biobin. This will give a 1 in 100 or 10-2 dilution of the original sample.
  5. In a similar manner prepare the rest of the dilution series up to and including 10-4.

 

 

 

 

 

 

Note: an animation of how to make 10 fold dilutions can be found on the HUB or by clicking the monitor below.

 

Preparation of a dilution series

 

 

 

 

3.3. The Viable count

 

 

Once the dilutions have been prepared a sample is taken as accurately as possible from each and plated onto a suitable medium. At a dilution in the dilution series there will be a countable number of bacterial cells per chosen amount of dilution. This magic figure is 30 – 300 colonies as these can be comfortably counted will give good results. Using the following instructions assess your sample for number of viable organisms using the techniques shown. You can use the same dilution series for each method.

 

 

3.3.1.

Figure 4.1. A diagrammatic representation of the pour plate method. Remember the bacterial sample must first be diluted using the method shown before any viable counting can occur

 

Viable counting method 1:  The spread plate

 

 

  1. Initially, label three of the prepared TSA plates by writing -2, -3 and -4 on the base of the dish along with your name lab number, session day and date. Place these in a row lid side up.
  2. Gently mix the contents of the -8 tube by swirling. Take a 200 ml Finn pipette set to 100 ml fitted with a small tip and withdraw 100 ml from the tube lift off the lid and dispense it on to the surface of the agar. Quickly, and using good aseptic technique repeat for the other plates and with the other dilutions. If you go in decreasing order of number i.e. 4 – 2 you can use the same tip.
  3. Finally using a disposable plastic spreader, starting at the -8 plate working quickly, gently spread the sample over the surface of the plate. Rotating the plate through 120 degrees after each spread before repeating so that the whole surface of the plate is covered as shown in figure 3.1.
  4. Invert the plate and incubate at 37oC for 24 hours.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.1. The spread plate technique. Add 100 ml of dilution to the plate and spread in tree directions to cover the whole plate using a disposable spreader.

 

Note: a video of how to perform the spread plate technique can be found on the HUB or by clicking the monitor below.

Preparation and counting of spread plates.

 

3.3.2. Viable counting method 2: The Miles and Misra method

  1. Using a non-water soluble ink pen, draw 4 sectors on the back of one of the prepared TSA plates, label these sectors 10-1 to 10-4 as shown in figure 3.2.
  2. Take a Finn pipette and fit a tip to deliver 20 µl. Mix the 10-4dilution and remove a 20µl sample. Holding the pipette vertically, with the tip about 1 cm above the surface of the agar, place the sample onto the agar in the sector labelled 10-4.
  3. Using the same Finn pipette and tip, repeat the procedure for the other dilutions in descending order of number. When completed, discard the tip into the biobin.
  4. Carefully push the plates towards the center of the bench where they will not be disturbed as the liquid soaks into the agar.
  1. When the liquid has soaked into the agar, invert the plate and incubate at 37oC for 24 hours.

 

 

 

20 µl samples of each dilution are placed onto the surface of the agar

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.2. Preparation of the Miles and Misra plate. You can use this diagram as a template to aid you in setting up the plate ready to add the 20 µl drops of each dilution to the surface of the agar in each segment. Remember to use the same tip going down the numbers.

 

 

 

 

Click here to see the video on the HUB

 

 

NOTE: After incubation your viability assay results will be provided via photographs posted on the teaching HUB

 

 

3.4. Interpretation of the viable counting experiment

 

 

  1. For the spread plate count all of the colonial bacterial growth (colonies) seen on the surface of the agar and record the number. This is easily done from the back of the plate by putting spot on each colony as you count them with a plate marking pen.
  2. For the Miles and Misra assay find a dilution segment where there are between 5 and 20 countable colonies, count them and record the number.

 

 

3.4.1. Calculate the CFU/ml from each assay (It’s very easy!)

 

 

To calculate the colony forming units per ml (CFU/ml) we need some information first.

 

  • You need the colony count
  • The amount of bacterial dilution you added to the assay initially
  • Dilution factor of the plate you counted

 

You then simply follow the process below

 

  1. Multiply the colony count by the amount need to get the quantity of dilution added to 1000 µl – For the spread plate this is 10, Miles and Misra 50 and for the pour plate 1.

 

  1. Then Multiply the adjusted count by the dilution factor expressed as a positive number

 

  1. The answer will be a big number. This expressed as an exponent or a ‘to the power of’ number see the example below.

 

Colony Count = 250

 

Amount plated =100 µl

 

Dilution plated =10 -7

 

 

250 x 10 = 2500

(This gets us to 1 ml of the dilution remember for Miles and Misra multiply by 50)

 

2500 x 10+7 = 2500,000,000,0 or expressed as 2.5 x 1010

(Don’t forget to change it to a positive number)

 

The final answer = 2.50 x 1010 cfu/ml

(Always to 1 or 2 decimal places)

 

 

 

  1. Carry out this process for each count from the example assays. A short power point on how to do carry out the count and cfu/ml calculation is available on the HUB

 

Click here to see power point slides on the HUB

 

 

 

 

3.4.2. Results from the 3 different viability assays you performed

 

 

Colony count and dilution in spread plate

 

 

CFU/ml of original E. coli sample was

 

 

 

Colony count and dilution in Miles and Misra

 

 

CFU/ml of original E. coli sample was

 

 

 

Please note. It is important to master this fundamental skill as there may be an examination question using this method.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3.5. Identification of Microbial pathogens

 

Often it is essential to be able to identify a bacterium precisely to the species level.

Many different ways of identifying bacteria have been developed. These range from simple biochemical tests to machines that can identify pathogenic bacterial or yeast species to strain level in a matter of hours. These may use variations of biochemical tests printed on cards but the most modern technology use the polymerase chain reaction (PCR) or analysis of the structure of the proteins that make up the cells themselves.

In this exercise you will use and interpret a variety of simple biochemical tests, combined with the Gram stain technique and observations of colony morphology and cellular arrangement to identify a mystery bacterial pathogen.

 

3.5.1. Identification of your mystery bacterial pathogen

 

You have been provided with:

  • A TSA plate containing colonies of the bacterial organism which you must identify as a dilution streak plate with a number or letter on.
  • A pre-prepared heat fixed smear of the same organism
  • Reagents and equipment to perform a Gram stain.
  • Reagents and equipment to perform a catalase test.
  • Reagents and equipment to perform an oxidase test.
  • Four pre-inoculated/incubated sugar fermentation biochemical tests in test tubes.
  • A disk diffusion plate corresponding to your unknown organism

 

You must perform all of the necessary observations, interpretations and tests to fill in the blanks in the identification tables for your organism and provide the identity of the pathogen by comparing your results to those provided.

 

 

 

 

 

 

 

3.5.2. The tests

 

Initially, record the number or letter on your streak plate and assess your plate for purity by looking to see if different colonies are present on the agar plate you have been given. Contamination sometime can get in around the edges of the plate. If you have different colonies on the plate you can carry on with the experiment but inform the staff and ensure you use a colony which is represented in the majority on the plate.

 

 

3.5.2.1. Morphological characters

 

Record the colonial morphology by examining a typical isolated colony on your streak plate. Remember to use the descriptive terminology used earlier in your practical sessions and that pigment production often shows by a change in the surrounding agar growth media.

 

You then need to perform a Gram stain on a heat fixed smear of the unknown bacteria using the method you have used previously. Record the Gram reaction, cellular morphology and cellular arrangement again using the correct terminology. Discard the slide into the disinfectant pot after use. Record the result in table 3.1.

 

3.5.2.2. The catalase test

 

Use a wire loop to remove some colony from the streak plate of your organism, and place it on a clean glass slide. Put a drop of hydrogen peroxide from the end of the bench onto the bacteria. Catalase positive bacteria cause the evolution of oxygen which is seen as a mass of bubbles. A slow reaction may be observed under the microscope using low power. If nothing has happened after 10 minutes the organism can be considered catalase negative. Discard the slide into the disinfectant pot after use. Record the result in table 3.1.

 

 

3.5.2.3. The oxidase test

 

Put a drop of Kovac’s oxidase reagent, which can be found at the end of the bench, onto a piece of filter paper and use a wire loop to smear some bacterial colony onto the area. A positive response is shown by an instant (within 10 seconds) purple colour being produced around where you have smeared the bacterial cells. Kovac’s reagent is light sensitive and will turn purple over time. Only an instant reaction is considered as positive. Discard the filter paper to disinfectant. Record the result in table 5.1.

 

 

3.5.2.4. Sugar Fermentation biochemical tests

 

Firstly, Check that growth has occurred, shown by turbidity (cloudiness in the liquid) after mixing, if there is no growth then the test is not valid. Fermentation of the sugar by the organism is shown by acid production causing the bromocresol purple pH indicator to turn yellow. This would be a positive result. Sugar fermentation products may also include gases which will collect in the small upside down test tube in the liquid which is called a Durham tube. The bubble must be sufficient to fill the curved end at the top of the tube. If fermentation has occurred record in table 5.1 whether this is -, + or + and gas. Record the result in table 3.1.

 

 

3.5.2.5. The Disk Diffusion assay

 

Measure the diameter of the zone of inhibition (no growth) of around each antibiotic disk on the surface of the agar plate. Resistance to the antibiotic on the disk is taken as a diameter less 10mm and susceptibility greater than or equal to than 10mm. Record these results for your unknown organism in table 3.2.

 

 

 

 

 

 

 

 

 

 

 

Name of bacterial species Cell shape Cell arrangement Gram reaction Catalase Oxidase Glucose fermentation Fructose

Fermentation

 

Lactose

Fermentation

Sucrose

Fermentation

Pseudomonas fluorescens Rod Single + +
Escherichia

coli

Rod Single + +

Gas

+

Gas

+

Gas

Staphylococcus aureus Cocci Irregular clusters + + + + + +
Bacillus megaterium Rod Pairs or short chains + +
Number or letter of unknown culture is:

 

ROD SINGLE + + + +

To identify the pathogen simply complete the blanks in the table below and compare your results to the diagnostic characteristics provided.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 3.1: Table of characteristics for interpretation of biochemical bacterial identification tests. Simply match the characteristics.

 

Name of bacterial species Penicillin

(P)

Ampicillin

(AMP)

Streptomycin

(S)

Chloramphenicol

(C)

Pseudomonas fluorescens R R S S
Escherichia

coli

R S S S
Staphylococcus aureus S S R S
Bacillus megaterium S S S S
Number or letter of unknown culture is:

 

       

 

 

 

 

Table: 3.2. Table of interpretive characteristics for interpretation of antibiotic disk diffusion assay of the unknown bacterial pathogen. NOTE: Resistance is taken as a diameter less 10mm and susceptibility greater than or equal to than 10mm.

 

 

 

Use the various characteristics to determine the identity of your unknown bacterial pathogen.

 

 

The identity of your organism is:

 

 

 

This practical session will provide the results necessary for you to complete the course work assessment for this module. Please see your module WOLF page for more details.

 

 

 

 

 

 

 

 

  • Formative questions:

 

  1. Describe the rationale behind how you made the decision as to the identity of your bacterial organism

 

 

 

 

  1. Why are the sugar fermentation tests different between each of the organisms?

 

 

 

 

 

 

  1. Why do E. coli cells produce gas during fermentation and the other species do not?