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How do you interpret this microbiology/ bacteriology research figure?

How do you interpret this microbiology/ bacteriology research figure?


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This is a figure from a research article that I am to do a senior presentation for. It is showing bacterial replication of an enteric bacteria population. The researchers hypothesize that the number of regions near the replication origin will be the highest point (peak) on the graph and the terminus region will be the lowest point because there are least copies of this region in a dividing species. The determine that the ratio of the peak to the trough will reflect the growth rate of the species. That all being said, I don't understand their use of color. The peak has several different colors that do not seem to match any of the examples on the right. Wondering if anyone can help me decipher this? Thanks!


The short answer is that it looks like the colors are meant to represent different, partial copies of the bacterial genome in the process of being synthesized.

When bacteria replicate, they initiate replication at the origins of their chromosomes. In fact, these origins can be replicated multiple times before it gets to the "end" of the bacterial chromosome (terminus). Looks like the stacking on the right figure shows this, with the colors just aiding in differentiating the partially replicated chromosomes. The coverage plot on the left shows how this might look based on read coverage from deep sequencing (again the colors just show different, partially replicated, chromosomes).


I think the figure has the answer. "High copy number near the origin". This is depicted in the graph by the spike at x = 4Mbp. The graph also shows that the number of reads is lowest at a point opposite the ori (2Mbp).

Mbp stands for Mega-base pairs, or 1 million nucleotides. Mbp in the graph refers to a point on the chromosome, with 4Mbp being at '12 oclock', the origin of replication(ori), and 2Mbp being at '6 oclock' opposite the ori. The researchers are measuring the number of DNA fragments(y-axis) that map to a certain position on the chromosome (x-axis). I am slightly color blind, but it seems to me that the colors generally match up. Also note that the reads that fall within the lower-most bracket on the right side of the x-axis span the entire x-axis. This is because these fragments map completely to the chromosome, and indeed they are labelled 'original genome'.

Hope that helps.


First, it would help if you could provide any more information on the figure (how it was made, is it data based or representative of a model?)

Given that there is little to go on, I can offer my two bits - and I hope it will help you get thinking about it - but…

It appears to me that these data are a snapshot of the replicating bacterial genomes isolated from many cells at a given timepoint. The right side of the figure shows a model of the (circular) genome in black with a number of smaller fragments of the genome caught partway through the replication process. The left side shows the same fragments stretched out in a linear representation.

The green fragment is the largest as it is nearly finished copying. Other colors show different sized fragments at different points in the copying process. It sounds like the graph is meant to relate the number of different sized fragments to growth rate (assuming you measure fragments from the same number of cells).

So, what does this tell you? (I think it would be helpful for you to think this out yourself form here and determine if you believe this is a good measure or not.)


Definitions:

Quantitative analysis
Analysis where a microbial count is determined

Qualitative analysis
Analysis where the presense or absence of an organism is determined

CFUs
Colony Forming Units

COA
Certificate of Analysis

This portion is a representative sample and will provide an accurate and comprehensive result.

When reporting a result where no organisms are detected, the reporting standard dictates that you cannot report zero, but that you need to report <1.

Since samples are diluted for testing purposes, this dilution also needs to be taken into account when reporting the result. For example, where no colonies are detected in a dilution of 1:10, the result would equal <10.

This result is the lowest result reportable where no organisms are detected.

Refer to the extract from ISO 7218, ‘Microbiology of Food and Animal Feeding Stuffs — General Rules for Microbiological Examinations’:

‘9.3.5.3.2 If the two dishes at the level of the test sample (liquid products) or of the initial suspension (other products) do not contain any colonies, express the result as follows:
less than 1 microorganism per millilitre (liquid products)
less than 1/d microorganisms per gram (other products)
(Where d is the dilution factor of the initial suspension)’

These results indicate that the microbial count reached the upper countable range of the test method. The counts are TNTC (Too Numerous to Count) and therefore are reported as more than (>) the upper countable range of the test.

Where counts exceed the recommended countable range, but are able to be counted, the result will be reported with an ‘E’ to indicate that the count is outside of the recommended countable range.

The reporting format of results is dictated by the ISO Standard 7218, ‘Microbiology of Food and Animal Feeding Stuffs — General Rules for Microbiological Examinations’.

This standard dictates the following:
In liquid samples, the lowest limit of detection for absence of growth is <1.

As a dilution of solid foodstuffs is made in order to test the sample, the lowest limit of detection for absence of growth is <10.

Since a representative portion of a sample is analysed, the results indicate that no growth was obtained in the sample portion tested.
A result of ‘no growth’ or ‘zero’ is therefore not scientifically accurate because the sample dilution is not reported, or taken into account.

The ‘E’ = Estimated
Most quantitative reference methods have a recommended range of reportable results. When counts outside of this range are reported, it is indicated using the symbol ‘E’. This count represents the actual colonies counted where the final count is outside of the recommended reporting range.

Example:
The countable range for TVC (Total Viable Count) is 30-300 CFUs/g per dilution. The results on the COA will read as follows for the following counts:
Average CFU count = 10 – Results = 10 E
Average CFU count = 350 – Results = 350 E
(For ease of interpretation, no dilution factor has been included in the example above)

Please note that we report all of our results as per international standard, ISO 7218, ‘Microbiology of Food and Animal Feeding Stuffs – General Rules for Microbiological Examinations’.

Please refer to point 9.3.5.3.1 and 9.3.5.3.2, wherein the following is stated with regards to calculation and reporting of results (see below):

‘9.3.5 Expression of results
9.3.5.3 Estimated counts
9.3.5.3.1 If the two dishes, at the level of the test sample (liquid products) or of the initial suspension (other products), contain less than 15 colonies, calculate the arithmetical mean y of the colonies counted on two dishes.
Express the result as follows:
for liquid products: estimated number of microorganisms per millilitre NE = y
for the other products: estimated number of microorganisms per gram NE = y/d
(where d is the dilution factor of the initial suspension).’

9.3.5.3.2 If the two dishes at the level of the test sample (liquid products) or of the initial suspension (other products) do not contain any colonies, express the result as follows:
less than 1 microorganism per millilitre (liquid products)
less than 1/d microorganisms per gram (other products)
(where d is the dilution factor of the initial suspension).’

Preliminary results indicate analyses which are complete. They may also show results which have not been completed, and are pending.

Preliminary results can be provided upon request of the client. They may be used by the client to establish further testing requirements. They may be used by the laboratory to convey results to the client before the final report can be issued.

When preliminary results are provided in the form of a Certificate of Analysis, they will not be signed. A signature indicates that the results and request have been verified by a technical signatory, and that the results are complete.

Certain reference methods stipulate that samples with presumptive positive results require further confirmation. Confirmation is therefore a obligation to complete the method, and report the final result as ‘Positive’ or ‘Detected’.

Results which have no presumptive colonies are complete according to the method. No further confirmation can be performed, and no additional result or charge for confirmation is therefore needed for these samples.

The results, and the additional cost associated with performing the confirmations are therefore separated.

NR indicates a non-reportable result, and will be indicated in the results column of the COA. Results may not be reportable for a number of reasons.

NR is reported when results obtained are not microbiologically sound, for example, the counts are not within laboratory acceptable limits or the food matrix affected the test media, resulting in colonies enumerated not portraying typical bacterial characteristics, or results.

In the case of NR, the cost for that particular analysis is removed and it is recommended to re-submit that particular sample for analysis so as to obtain conclusive results.


Scope & Mission

Frontiers in Microbiology is a leading journal in its field, publishing rigorously peer-reviewed research across the entire spectrum of microbiology. Field Chief Editor Martin G. Klotz at Washington State University is supported by an outstanding Editorial Board of international researchers. This multidisciplinary open-access journal is at the forefront of disseminating and communicating scientific knowledge and impactful discoveries to researchers, academics, clinicians and the public worldwide.

As we are taking much better account of the unseen majority of life, unravel the biogeochemical processes that microbes facilitate, thereby making planet Earth habitable for all forms of life as we increasingly identify the rules by which microorganisms interact with co-evolving viruses and macroorganisms in health and disease and as we find more and better strategies to mitigate the detrimental effects of anthropogenic activities on the abundance, diversity, distribution and activity of microbial communities, Frontiers in Microbiology will be the 21st century approach to communicate all this progress to both the specialist and a wider audience of readers in the field.

Frontiers in Microbiology is a member of the Committee on Publication Ethics.


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Microbiology Unknown Lab Report by Taylor Autry

In this paper I will discuss the processes of how I came to find my two unknown bacteria. This will be a vital task to take with me into my profession for many reasons. In the medical field bacteria and infections of different kinds are the core of the practice. These bacteria must be able to be identified in order to treat patients properly, efficiently and safely.


Materials and Methods:

The unknown number 123 handed out by the Professor on March 20, 2014 contained both a gram positive bacteria and a gram negative bacteria. At this point everything that had been learned in microbiology lab and that had been explained in our lab manual (1) was put into action. The first step to figuring out the unknowns, was to separate the two bacteria. In order to do this, a nutrient agar plate was used. The streak method was used to spread the bacteria across the nutrient agar in hopes of isolating a pure culture of one of the bacteria. In order to do the streak method, an inoculating loop was sterilized with a Bunsen burner and put into the unknown specimen. After removal with bacteria on the loop, the quadrant streak method was used. The streak plate was then incubated at 37 degrees Celsius for 48 hours. Upon returning and observing the streak plate, there was an abundance of green across the plate. There was only one colony that was apparent. After observation, a sample was taken from the isolated colony on the streak plate and another streak plate was done with that, trying to further isolate the colonies. As well as using the quadrant method to further isolate the colonies, a sample was taken from the best colony on the original streak and gram stained. The gram stain procedure was performed as directed in the lab manual (1). The gram stain showed a result of red, gram negative rods. To decipher between which biochemical tests to perform, the gram positive and negative tables handed out by the Professor, were referred to. From previous biochemical tests done in the semester, Pseudomonas aeruginosa was already suspected because of the green pigment of the original streak plate. Upon reviewing the identification tables, the deciding biochemical test was the Casein test which tests for the production of the enzyme casease to break down the milk protein casein. The milk agar was incubated at 37 degrees Celsius for 48 hours. After observation there was a clear positive result, which showed the bacteria produced casease. After confirming the gram negative bacteria, the process of isolating the gram positive bacteria began. Returning to the original unknown stock 123, a quadrant streak plate was done with a sterilized inoculating look on a mannitol salt agar, which inhibits the growth of gram negative bacteria. This MSA plate was incubated at 37 degrees Celsius for 48 hours. Upon return and observation, the MSA did not yield a good isolated colony. Professor Snaric advised to do another MSA agar from the original stock, but this time with a sterile swab instead of an inoculating loop. This test was also incubated at 37 degrees Celsius for 48 hours and returned a good isolated colony. A sample was taken from this colony and transferred to a nutrient broth agar to further isolate it. After incubation, this nutrient agar had great results with many isolated colonies. A sample was taken from the isolated nutrient agar and a gram stain was done as directed by the lab manual (1). The gram stain showed clear purple gram positive cocci. After getting a good gram stain the identification tables were referred to in order to choose between appropriate biochemical tests.

A nitrate test was performed in order to detect if the bacteria was able to reduce nitrate into nitrate or some further reduced form. Then a urea test was performed to check for the production of urease. All of the biochemical tests performed were explained, in the lab manual provided by Professor (1) and were practiced earlier in the semester. Tables 1 and 2 lists the tests, purposes, reagents used and the results of each test.

The following tests were performed on the Gram Negative bacteria:

The Following Tests were performed on the Gram Positive bacteria:

The first test performed on the gram negative bacteria, was a Casein Test. This test gave a positive result turning a brown color, meaning the gram negative bacteria produced the enzyme casease in order to break down the milk protein casein. This was the only test necessary to determine the unknown gram negative bacteria in unknown stock 123.

Table 1: Tests and Results for Gram Negative Bacteria

The first test performed on the gram positive bacteria was the Nitrate Test which turned red after adding reagents giving a positive result meaning the bacteria reduced nitrate into nitrite or something further. Following the nitrate test was the Urea Test to determine if the bacteria produced urease. This gave a positive result showing a hot pink broth, meaning the bacteria did produce urease.

Table 2: Tests and Results for Gram Positive Bacteria


Discussion/Conclusion

The unknown #123 contained two different specimen of bacteria, one being a gram positive bacteria and one being a gram negative bacteria. The first unknown in #123 found to be a gram negative bacteria, was identified as Pseudomonas aeruginosa. This identification was reached by only one biochemical test and close observation. A gram stain was done originally and found red rods identifying the bacteria as gram negative. The streak plate that was originally done, showed a heavy green pigment, which is a main characteristic of Pseudomonas auruginosa. The next test performed was a Casein Test which showed a clear positive result. The only gram negative bacteria that shows a positive result on the Casein Test is Pseudomonas aeruginosa, therefore correctly confirming the unknown gram negative bacteria.

The second unknown bacteria was identified as a gram positive bacteria with a coccus shape. This immediately ruled two of the gram positive bacteria Bacillus cerus and Bacillus subtilis. This left three bacteria that the second unknown could be Staphylococcus aureus, Staphylococcus epidermidis, or Enterococcus faecalis. The next test performed was a Nitrate Test which gave a positive result. The two bacteria that give a positive result for this test are Staphylococcus aureus and Staphylococcus epidermidis. The second test performed was a Urea Test which also gave a clear positive result confirming that unknown gram positive bacteria as Staphylococcus epidermidis because Staphylococcus aureus gives a negative result. Consultation with the Professor, confirmed the two unknown bacteria correctly as Pseudomonas aeruginosa and Staphylococcus epidermidis. The only problem that was encountered appeared when trying to isolate the gram positive bacteria. The gram negative bacteria in unknown #123 is a very aggressive bacteria that makes the growth of a gram positive bacteria difficult. Even on an MSA agar, it took a few tries and several isolations to get the gram positive to successfully grow. After successful isolation of both bacteria, there were no more issues encountered in identifying either.

Pseudomonas aeruginosa is a gram negative rod shaped bacteria that was first discovered in 1882 by a pharmacists named Carle Gessard (2). His study picked up on the unique blue-green pigmentation of P. aeruginosa. This bacteria can catalyze in many environments which makes it very common and found almost everywhere such as soil, water, humans, plants, sewage and hospitals. P. aeruginosa is what is called an opportunistic human pathogen, because it rarely affects a healthy individual. This bacteria more so affects individuals with compromised immune systems the most common being those with cystic fibrosis, cancer, or AIDS. (2) This particular bacteria is so dangerous and pathogenic that it infects up to two thirds of critically ill patients in the hospital and is a leading pathogen in most medical centers with a mortality rate of 40-60% (2). It is most dangerous to cystic fibrosis patients and is involved and complicates 90% of cystic fibrosis deaths. It builds resistance against a lot of antibiotics and even chemotherapeutic agents. “P. aeruginosa is a facultative aerobe its preferred metabolism is respiration.” (MicrobeWiki) People that are most at risk for infection of P. aeruginosa are those in hospital settings, especially ones on breathing machines or with catheters (3). Although some cases have been caused by swimming in pools or hot tubs with incorrect levels of chlorine. To avoid the spread and contamination of P. aeruginosa nurses and health care professionals should be sure to use aseptic technique because the bacteria is mostly spread through contaminated equipment and professionals hands. P. aeruginosa is quickly on its way to becoming resistant to antibiotics and becoming increasingly difficult to treat. Selecting the correct antibiotic for this particular bacteria is especially important.


The Future

Research suggests that efforts to make a cleaner environment, free from bacteria, are contributing to the rise in obesity, cancer, and heart disease. [6] Experts are trying to figure out how “probiotics” (foods like yogurt with active cultures and dietary supplements that contain live bacteria) can improve our health. Research is underway so that in the future, specific bacteria may be prescribed as individually tailored treatments for patients.

Our immune system needs the right combination of bacteria so we can stay healthy and rely less on medications. Antibiotics remain a powerful tool to keep us healthy but shouldn’t be used when they aren’t needed. The more we learn, the more we appreciate the power of the bugs inside of us—to heal and not just to do harm.

All NCHR articles are reviewed and approved by Dr. Diana Zuckerman and other senior staff.


Positive control: Clostridium perfringens (ATCC 13124)
Negative control: Escherichia coli (ATCC 25922)

  1. Collin County Community College District
  2. Portland Community College
  3. ASM Microbe Library: Endospore Stain Protocol
  4. Austin Community College
  5. Western Michigan University
  6. Sigma-Aldrich Chemie GmbH
  7. Bacteriological Analytical Manual 8th ed., Revision A (1998)
  8. H.J. Conn’s Biological Stains, 9th ed. by R.D. Lillie (1977)
  9. Fall 2011, Jackie Reynolds, Richland College, BIOL 2421
  10. Microbe Online
  11. Wikipedia

3 thoughts on &ldquoEndospore Staining- Principle, Reagents, Procedure and Result&rdquo

How is this staining procedure similar to spore staining technique

I am currently taking a microbiology course at the local community college. I have an unknown only known to my professor. I performed 3 staining procedures and determined that my unknown is gram-positive cocci, non-acid fast, endospore former. I thought it might be a Mycobacterium. From the information I provided, is that correct? Or is more information needed?

Hello Sagar Aryal,
I have a doubt….
Before I performe endospore stain I believe that I need that the bacteria be on endospore phase….How I do that?


Eligibility & Admission

You can take admission in bachelor courses after passing your 12 th class with PCB subjects. Some colleges consider 50% marks in PCB subjects.

For admission to PG courses, it is necessary to hold a bachelors degree (B.Sc.) in the related field. Some universities organize their own entrance exams for screening candidates for admissions. Candidates can appear in NEET PG 2021, AIIMS PG 2021 and JIPMER PG 2021 for microbiology courses.

Best colleges to pursue Microbiology are:

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Skills Required for a Microbiologist:

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