Basic microbiology: gram positive and gram negative organisms

Gram staining is one of the basic staining technique used in microbiology laboratories. It was discovered by Hans Christian Gram in 1884. This technique uses bacterial property of having peptidoglycan in their cell wall.

Hence, bacteria lacking a cell wall cannot be stained using this method – e.g. Mycoplasma.

Gram staining process
1. Make a smear on a glass slide using bacterial colony, heat fix.
2. Put a crystal violet (blue dye)
3. Put iodine (to fix the dye)
4. Decolourise using ethanol/acetone – If the bacteria have large amount of peptidoglycan in their cell wall, i.e. they have a thick cell wall, they will retain the blue dye, these are gram positive organisms.
5. Put a red/pink stain (safranin, neutral red etc) – bacteria with a thin cell wall with small amount of peptidoglycan, would have lost the blue stain in the previous stage. These will appear red, these are gram negative.

Here is a video showing how to do gram stain
This is national standard (SMI) for gram stain (and other staining methods) – if you are interested

So gram positive organisms are blue, gram negatives are red.
Occasionally it becomes difficult in some bacteria as they may stain inconsistently – we call them gram variable (we would exclude them from this discussion).

We classify bacteria based on their shape and how they appear in relation to each other.
Common shapes are –
round = coccus (pl. cocci)
long rod like = bacillus (pl. bacilli)
(There is a genus of bacteria called Bacillus – do not confuse these two terms. Genus Bacillus is indeed a bacillus shaped bacteria but, there are other bacteria with this shape – E coli, Klebsiella, Clostridium etc).
However, some bacteria may appear somewhat in the middle of these two categories – they are called coccobacilli.

These bacteria may form a particular type of configuration, which help us to make an educated guess of their genus.  They may form cluster, chains, pairs, Chinese letter appearance. However, it is difficult, if not impossible, to predict the species of gram stain only.

Here are some examples:
Gram positive cocci in cluster:
Staphylococcus aureus, 
Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus simulans etc.
Gram positive cocci in chains
Streptococcus pyogenes (Group A Strep), Streptococcus agalactiae (Group B Strep), Streptococcus dysgalactiae (Group C/G Strep), Streptococcus gallolyticus (Group D Strep), Streptococcus anginosus (milleri) group, 
Gram positive cocci in pairs:
Pneumococcus (Streptococcus pneumoniae), Streptococcus from mouth flora (These are alpha haemolytic Strep - Strep salivarius, Strep sanguinis, Strep oralis, Strep mitis, Strep mutans etc).

Enterococcus (Enterococcus faecalis, E faecium etc) usually present as short chain or in pairs
Gram positive rod/bacilli
Bacillus cereus, Bacillus anthracis (causes anthrax), Clostridium difficile, Clostridium tetany (causes tetanus), Clostridium botulinum, Clostridium perfringens, Clostridium novyi (causes gas gangrene)
Listeria (meningitis, neonatal infection etc)
Corynebacterium sp. (causes diphtheria but also some species live on our skin and normally harmless)

Gram negative cocci:
Acinetobacter, Moraxella - they can often be seen as coccobacilli

Neisseria meningitidis (diplococci - two coccus together), Neisseria gonorrhoea (diplococci).
Gram negative rod/bacilli
Escherichia coli, Klebsiella, Enterobacter, Serratia, Salmonella, Shigella, Yersinia, Proteus, Morganella

Some gram negative bacilli may have typical appearance-
Campylobacter (curved gram negative rod)

Vibrio e.g Vibrio cholerae (comma shaped)

Fusobacterium nucleatum (an anaeorobic bacteria from the mouth) - needle shaped
Yeast- like Candida, appears gram positive.



List of resources

This is a list to help you prepare for the examination, but I do not say that this is a comprehensive list. There are many other excellent resources out there to help you. Please let me know if you feel there is something which should be mentioned here.
 Many of these books are costly and you may want to check your departmental/institutional library first.
  1. Curriculum – Most important resource. Go throught the curriculum and do a gap analysis. Check what are the areas you feel you need to read more. You can develop a traffic light system to help you (green – you are confident, amber – you need to read more, red – you must read about this topic. For example ‘typing in microbiology’ How confident are you?)
  2. A standard textbook: Patrick Murray/ Greenwood.
  3. Oxford Handbook of Infectious Diseases and Microbiology
  4. Microbiology & Infectious Diseases Flashcards, Third Edition (Lange Flashcards)
  5. The emphasis on clinical aspect of the infection has increased now. Hence, I believe preparing from a USMLE book (infection related questions) will be helpful. However, please be aware that this exam is for the UK, so you have to think about the UK scenario when you are preparing. (For example – the coccidioidomycosis is endemic in some parts of  the USA but not in the UK, MRSA is more prevalent in the USA,  teicoplanin is not available in the USA, Group B universal screening in pregnancy is not done in the UK etc).
  6. Critical appraisal:
    Book –How to Read a Paper: The Basics of Evidence-Based Medicine: Trisha Greenhalgh
    Practice writing abstract. Make a list of different statistical terms (e.g Sensitivity, specificity, NNT) and learn how to apply them.
  7. Vaccine: Greenbook
  8. Greenbook MCQ
  9. SOP: National SMI
  10. Sensitivity testing – EUCAST
  11. Mycology: Identification of Pathogenic Fungi  (- A book to help with mycology identification. If you have any other resource and comfortable with it – please use that. )
    Dividing the fungi based on clinical features might help. For example, if the sample is an ear swab – you should be able to think what are the common fungus that we can isolate from there. Make sure you are familiar with microscopy, germ tube test, API, E-test, corn meal agar. Learn different methods of identification and sensitivity. You must read about clinically relevant fungi and antifungal drugs.
  12. Virology: Read the virology from any standard textbook. I read Kudesia’s Clinical and Diagnostic Virology, but I couldn’t find any updated version after 2009. I do not recommend using a reference book like Fields Virology as a textbook (it is not time effective).
    I suggest you collect a list of common virology problems (e.g. management of rash contact in pregnancy) and prepare answers for those scenarios. Ask your virologist for help.
  13. Parasitology: Usually short answer questions, spotters etc. Please remember UK not being a tropical country we do not see a lot of parasitology. You need to balance your effort and gain here. I also found it useful to keep google image open when I read parasitology. Search the image of whatever you are reading. It will help you if you get a spotter.
  14. Cowan and Steel’s Manual for the Identification of Medical Bacteria (Reference, most likely you will get it in your laboratory)
  15. Health technical memoranda (relevant ones only – water, ventilation, decontamination)
  16. Subscribe to Public Health England (PHE) RSS feed or
    Subscribe to Health Protection report
  17. Journals – Read UK microbiology journals
    Journal of Antimicrobial Chemotherapy
    The Journal of Hospital Infection
    Journal of Infection
    The Lancet Infectious diseases
  18. Guideline from different UK organisations – NICE, BTS, BSAC, BASHH, BHIVA, CHIVA, HIS, BIA, SIGN, IPS,  RCOG, BOA, BSR etc.
  19. Infection control: Check NHS Trust guidelines – many of the NHS trust guidelines are available in the internet. If you are from the UK – read your trust infection control and antibiotic guideline. If you are in a DGH – find out the guidelines of a university hospital, as you may not find all the guidelines in a DGH – like transplant related guidelines.

You can also supplement your knowledge from an infection control book (some of them are very costly and old – choose a new one, if you can)
Communicable Disease Control and Health Protection Handbook (2012)

Manual of Infection Prevention and Control

Ayliffe’s Control of Healthcare-Associated Infection Fifth Edition: A Practical Handbook (2009)


Unfortunately I am not aware of any preparatory course for part 2 FRCPath Microbiology. (If you are aware of any -please let me know).

For UK trainees local hospitals might organise a mock test for you. I recommend you speak to your ES and TPD.

Past questions:

RCPath do not allow storing of the past papers, but you can look at some sample papers here.

You should speak to trainees who has recently appeared in the examination for guidance.

Commonly asked questions about FRCPath Microbiology

Here are some topics that I get asked frequently. Many of these questions are answered in the RCPath website - please read this first -RCPath FAQ for the examination .

Please note I am neither involved in the examination process nor hold any position in RCPath. These answers are from my own experience.
How much time you should give yourself to prepare?

It is difficult to say how long you need as it very much depend upon the you – your method and speed of study, whether you are preparing while working or not, whether you are trained in the UK or familiar with UK system etc.

For UK trainees – I would suggest you discuss with your educational supervisor. He is the best person to advise you if you are ready.

For International trainees – if you have already done post-grad, probably you have enough knowledge of Microbiology. However, you need to get used to or learn the UK system. You might need 6 months least, if not a year, to prepare for either part of FRCPath.

Collect your materials first, then assess how much time you might need to cover that.

Is there any syllabus/curriculum?

RCPath provides the trainees with the curriculum.

Is there any MCQ book/Question bank available for the examination.

I have not seen any book specifically written for FRCPath examination. The questions are often based on clinical scenario. So if you find similar questions in any book (USMLE prep), please use that.  RCPath provided us with some sample papers.

You should also speak to the trainees who has recently appeared in the examination for guidance.

My understanding is that the FRCPath part 1 looks for a breadth of knowledge rather than depth. For Part 2, however you need an extensive knowledge of all aspects of microbiology and infection (and also areas like infection control, decontamination etc.) as you will be expected to work unsupervised as a consultant soon.

Resources for FRCPath part 2 by Dr Katherine Watson

Dr Katherine Watson, StR, Microbiology has kindly let us know the resources that she used to prepare for FRCPath part 2.
Thanks Katherine.
Here are some of the resources that I used in preparation for FRCPath part 2, April 2016 Bristol. One of the most useful things to do is speak to anyone you know who has already sat the exam and ask for all their past questions and tips.
Mycology – A Canadian microbiologist’s blog, has some really great photos of fungi, very useful for familiarising yourself with organisms for spotters and the practical. I printed quite a few out and stuck them around the house, after seeing them every day I felt confident at quickly recognising organisms.– Website set up by the University of Adelaide, lots of useful mycology information, the mould virtual assessment section is particularly good for testing yourself.– Suggest read the EORTC/MSG definitions of invasive fungal disease. – Recent review article published in the Lancet, update on topical issues surrounding diagnosis and investigation of invasive fungal infection that can come up in many sections of the exam.$FILE/Etest%20AFST%20Reading%20Chart.pdf – Useful pictures and guidance on how to interpret etests for fungi, useful to have in the lab folder.
Parasitology – CDC website has 18 years’ worth of monthly case study quizzes and A-Z of parasites, excellent practice for spotters, this is the resource I used for most of my parasitology revision.
The Green Book! Suggest reading this a few times, the virology paper is only an hour long, but the questions were all very specific questions that you either knew or you didn’t, for example, we had to give dosing schedules for Hepatitis B and rabies and specific regimen for HIV post exposure prophylaxis. Thankfully, we didn’t get any questions about the new Hepatitis C drugs. – Screening for Hepatitis E in blood products is a topical issue that we got asked about in the virology paper. Make sure to read all the latest guidance on the PHE website about the Zika virus, this is very topical so likely to come up in the next exam. We had to write a short note on the laboratory diagnosis of Zika virus.
Bacteriology – Quite time consuming, but I suggest reading all of the SMIs. I printed the flow charts from the identification section to put in my practical folder. I would suggest learning by heart the 2 SMIs under the protocol section which cover identification of ESBLs and CPEs as there were lots of questions and spotters on these topics.
Also, don’t forget to read the quality-related guidance, you can pick up useful information to include the essay question if it is anything about a laboratory service and also in my exam one of the groups of 4 short notes were all about laboratory quality assurance.
Look out for new SMIs, for example, in the autumn 2015 exam there were questions relating to the new ectoparasite SMI. – I would suggest printing out the BSAC endocarditis guidelines for the lab folder, so if one of the cases is about endocarditis you will have the guidelines to refer to for treatment advice.
Infection Control and Public Health
PHE website has some really good training slides for healthcare professionals with up-dates on specific vaccines, would definitely recommend reading since the vaccination programme seems to have changed quite a bit in recent years.
Must read documents for infection control of CPE
If you have time before the exam I would suggest completing Dundee University’s 6 week online course on antimicrobial stewardship, which is such a topical issue that can come up in the exam. You can sign up for free:
General – Another great website for practicing spotters, clinical cases with high quality pictures of clinical conditions, radiology, histology and organisms then multiple choice questions on diagnosis. You have to sign up to the website, but it’s free to do so. – Federation of Infectious Diseases Societies of Southern Africa website, has a case of the month section where you can guess the diagnosis, quite a lot of rare tropical infections. – American Academy of Dermatology, has a number of quizzes including some on infectious causes of rashes.
iTunes U – If you search microbiology or virology you can find some series of lectures from American universities, some good basic parasitology and mycology lecturers that you can listen to when walking or driving to work. -A well written article advising how to write an abstract, which is something that you always have to do for each of the critical appraisals. I suggest reading journal articles with the abstract covered and practice writing abstracts, pay careful attention to word counts, you are normally given a 250 word limit.
The Doctor’s Guide to Critical Appraisal Paperback – 14 Jul 2006 by N. Gosall, G. Gosall – I am terrible at critical appraisal and read a lot of books to try to improve, this was the one I thought was most useful with straightforward explanations of statistical tests and study designs. Other trainees told me that the written paper is where most people fail the exam, I have to agree and would definitely recommend doing a lot of preparation for critical appraisal. I read this book and also Trisha Greenhalgh’s How to Read a Paper: The Basics of Evidence-Based Medicine several times.

New format of part 2 FRCPath Microbiology examination

Source: RCPath website

From Autumn 2017 onwards, the format of the FRCPath Part 2 Medical Microbiology examination has changed. The changes, which have been approved by the General Medical Council, are designed to make the exam more accurately reflect the role of the consultant microbiologist in modern NHS practice; to be in line with new, revised curricula in Medical Microbiology specialty training; and to strengthen the reliability of the examination.

The new-format examination will last two days and be structured as follows:

Structured essay paper and Short-Answer Questions (SAQs): 2 ½ hours

1 essay (from a choice of 2) followed by 10 SAQs.

SAQs to be based on clinical and laboratory scenarios and to include:

  • Data interpretation
  • Report validation
  • Laboratory management, including quality assurance, laboratory error/incidents, lab safety
  • Clinical investigation and management
  • Infection prevention and control
  • Anti-microbial stewardship

Critical Appraisal Paper and SAQs: 2 ½ hours

1 journal article to critically appraise (from a choice of 2) followed by 10 SAQs (as described above).

Practical Scenarios: 3 ½ hours

  • A series of images, data and other laboratory-based material will be presented with clinical scenarios for which candidates provide interpretation and construct management plans.
  • Clinical scenarios and laboratory material evolve with additional information during the exam to simulate day-to-day clinical practice

Objective Structured Practical Examination (OSPE): 3 hours

At least 15 stations.