Clinical Microbiology 2, Study notes of Medical Microbiology

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LIST OF CONTENTS

• MLT 117 – CLINICAL MICROBIOLOGY
• CHAPTER ONE: ISOLATION OF BACTERIA………………........
• CHAPTER TWO: BACTERIA IDENTIFICATION………………...
• CHAPTER THREE: ANTIBIOTICS STUDIES OF BACTERIA…...
• CHAPTER FOUR: IDENTIFICATION OF FUNGUS……………...
• CHAPTER FIVE: IDENTIFICATION OF PARASITES …………...

Some types of specimen are normally sterile e.g. blood, CSF. These samples are usually obtained via a percutaneous route with needle and syringe, using appropriate skin disinfection and an aseptic technique. The culture of bacteria from such specimens is usually indicative of definite infection except if they are skin contaminants (bacteria inhabitants of normal skin). Fig 1: Universal container. In contrast, many microbiological specimens are obtained from non- sterile sites e.g. vaginal or throat swabs, urine sample, stool sample. Such samples often contain bacteria of no clinical relevance in addition to possible pathogens, making the interpretation of culture results more difficult. In general it is preferable to send samples from sterile sites if available. It is preferred to obtain the samples for bacteriological culture before antibiotic therapy is started. This maximizes the sensitivity of the investigations and reduces false-negative results. Similarly, samples of tissue or pus are preferred over swabs, to maximize the recovery of bacteria in the laboratory. Specimens must be accurately labelled and accompanied by a properly completed requisition form, indicating the nature of the specimen, the date of sample collection, relevant clinical information, the investigations required, and details of antibiotic therapy, if any.

5 This allows the laboratory to perform the correct range of tests, and helps in the interpretation of results and reporting. Along with clinical specimens, medical microbiology laboratories also process samples of food, water and other environmental samples (e.g. air sampling from operating theatres) as part of infection control procedures. High-risk samples Certain bacterial infections are a particular hazard to laboratory staff, and specimens that might contain these pathogens should be labelled as ‘high risk’ to allow for additional safety measures if necessary. For example - blood cultures from suspected typhoid (Salmonella typhi) or brucellosis ( Brucella species), and samples from suspected Mycobacterium tuberculosis. Preservation and Transport of specimen Most specimens are sent to the laboratory in sterile universal containers. Swabs are placed in a suitable transport medium (eg. charcoal medium) otherwise it leads to false negative reporting. Fig 2: Charcoal laden transport media Specimens should be transported as soon as possible to the laboratory. In case a delay is anticipated the specimen should be stored at 4° C. Immediate transport is necessary in order to: (i) Preserve the viability of the ‘delicate’ bacteria, such as Streptococcus pneumoniae or Haemophilus influenzae (delays in processing can cause false-negative culture results); (ii) Minimize the multiplication of bacteria (e.g. coliforms) within specimensbefore they reach the laboratory. In particular urine and other specimens that utilize a semiquantitative culture technique for

7 is performed from the deposit after the supernatant is decanted. This helps increase the sensitivity of both microscopy and culture. Ziehl-Neelsen (ZN) stain is used to demonstrate the presence of Mycobacteria. Mycobacteria can also be visualized using the fluorescent dye auramine and a fluorescence microscope. Direct immunofluorescence is employed to detect certain pathogens (e.g. Legionella, Pneumocystis ) using specific antibodies conjugated to a fluorescent dye. Another microscopic technique is dark ground microscopy. This is mainly used to detect the thin spirochaetal cells of Treponema pallidum (syphilis bacteria).

INTEXT QUESTIONS

1. Specimens that contain pathogens which are hazardous to laboratory staff should be labeled as ..................
2. Swabs are sent to laboratory in .................. medium
3. If delay is anticipated in transporting the specimen, it should be stored at .................. temperature
4. .................. gives an indication of bacteria present in the sample
5. .................. stain is used in demonstration of mycobacteria
   6. .................. microscopy is used to detect syphilis organism

METHODS OF ISOLATION OF BACTERIA

Methods of isolation of bacteria can be broadly classified into two: Culture methods: On Solid media, On Liquid media, Automated systems Non-culture methods Culture methods The first requirement for physically isolating a bacterium is that it can be cultured in the laboratory. This requires knowledge of optimal temperature for growth, optimal oxygen requirements, and optimal nutritional needs. The specimens received in the laboratory are plated on the culture media. The appropriate culture media is selected depending upon the bacteria suspected. The following precautions need to be taken into consideration when the culture methods are processed Optimal atmospheric conditions Optimal temperature Growth requirement of the bacteria Atmospheric conditions : Colonies of bacteria are usually large enough to identify after 18– 24 hours of incubation (usually at 37°C), but for some bacteria longer incubation times are required (from 2 days to several weeks). Culture plates are incubated (1) in air, (2) in air with added carbon dioxide (5%), (3) anaerobically (without oxygen) or (4) micro-aerophilically (a trace of oxygen) according to the requirements of the different types of bacteria that may be present in specimens. In case of Mycobacteria especially the scotochromogen the culture bottles are placed in dark or the bottles are covered with black paper and kept for incubation at 37°C. Temperature: Most of the bacteria requires a temperature of 37°C for optimal growth. This temperature is provided placing the inoculated culture plates in the incubator set at 37°C temperature.

Different bacteria produce different but characteristic colonies, allowing for early presumptive identification and easy identification of mixed cultures. There are many different types of culture media. Agar is used as the gelling agent to which is added a variety of nutrients (e.g. blood, peptone and sugars) and other factors (e.g. buffers, salts and indicators). Some culture media are nonselective (e.g. blood agar, nutrient agar) and these will grow a wide variety of bacteria. While some e.g. MacConkey agar are more selective (in this case through the addition of bile salts selecting for the ‘biletolerant’ bacteria found in the large intestine such as Escherichia coli and Enterococcus faecalis ). MacConkey agar also contains lactose and an indicator system that identifies lactose- fermenting coliforms (e.g. Escherichia coli, Klebsiella ) from lactose-non fermenting coliforms (e.g. Morganella Salmonella ). Media can be made even more selective by the addition of antibiotics or other inhibitory substances, and sophisticated indicator systems can allow for the easy detection of defined bacteria from mixed populations. Method of inoculating the solid culture media Method used for inoculating the solid media depends upon the purpose of inoculation- whether to have isolated colonies or to know the bacterial load of the sample (quantitative analysis). For obtaining the isolated colonies streaking method is used, the most common method of inoculating an agar plate is streaking. Fig 6: Streaking method

11 Streak plates

1. A small amount of sample is placed on the side of the agar plate (either with a swab, or as a drop from an inoculating loop).
2. A sterile loop is then used to spread the bacteria out in one direction fromthe initial site of inoculation. This is done by moving the loop from side to side, passing through the initial site.
3. The loop is then sterilised (by flaming) again and the first streaks are thenspread out themselves.
4. This is repeated 2-3 times, moving around the agar plate as shown in the figure. In this method single bacterial cells get isolated by the streaking, and when the plate is incubated, forming discrete colonies that will have started from just one bacterium each. For quantitative analysis or semi quantitative analysis of the sample for example in case of urinary tract infection. In fact E. coli is implicated as the causative organism in urinary tract infection only if there are

10^5 Colony forming units per millilitre of urine. The method of inoculating the solid culture media is as shown in the figure. Fig 7: Inoculation methods

13 Fig 10: Liquid media Automated system Automated blood culture systems eg. BACTEC, BacteAlert utilize liquid culture. Bacterial growth may be detected by a variety of methods (e.g. detection of bacterial CO 2 production). Fig 11: Bactec

Fig 12: Bactec Automated liquid culture systems are also available for the culture of Mycobacteria, and similar technology can be used to automate sensitivity The advantage of automated system are Rapidity : they aid in faster growth of bacteria. Thus less time consuming. The incidence of contamination during the processing of sample are minimised Real time monitoring of the growth One of the main limitations is the commercial viability. Non culture methods Isolation of bacteria can also be carried out by non-culture methods. In particular the more advanced Amplification techniques like Polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA) are being used in clinical laboratories for isolation and identification of bacteria. The following are some of the factors that are considered in interpreting bacteriological culture results:  type of specimen  any delays in processing  types of bacteria recovered

 Don’t leave your plate open too long or extra bacteria from the environment will fall into your plate.  Do not be disappointed if you do not get isolated colonies on your first try. This is a difficult procedure. ESSAY QUESTIONS

1. What is the need for isolation of bacteria?
   2. Describe in brief various steps involved in the isolation of bacteria.
   3. What is difference between blood agar and chocolate agar
   4. Explain the term selective and non-selective media with proper examples.
   5. Draw a labeled diagram of inoculation of solid culture media for isolation of bacteria.
   6. Draw a labeled diagram for inoculation of solid media for processing the urine sample of a patient suspected of urinary tract infection.
   7. Describe in brief the advantages and the limitation of use of liquid culturemedia for isolation of bacteria.
   8. Mention the advantages and the disadvantages of automated system for isolation of bacteria.
   9. Name some non culture methods for isolation of bacteria

17

ANSWERS TO INTEXT QUESTIONS

1. High-risk
2. Charcoal
3. 4 degree Celcius
4. Gram stain
5. Ziehl-Neelson
6. Dark Ground

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1. Direct culture & Non-culture
2. Factor V & Factor X

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1. Agar
2. Non-selective
3. MacConkey
4. Streaking
5. Broth
6. Polymerase Chain Reaction, Ligase Chain Reaction, Nucleic Acid Sequence Based Amplification

19 Gram negative bacteria can be either cocci or bacilli. Gram negative pathogenic bacteria commonly encountered are E.coli, Klebsiella, Salmonella spp, shigella, etc

2. Albert staining: is performed in case if one suspects a Corynebacterium spp.
3. Acid fast staining: is performed in cases suspected of Mycobacterial infection. Eg. Tuberculosis, leprosy, etc.
4. Special staining is necessary in case of spirochetes and other organisms.

INTEXT QUESTIONS 11.

1. ......................... of bacteria is the important step in identification of bacteria
2. Gram stain differentiates bacteria as ......................... & .........................
3. ......................... staining is used in Identification of Corynebacterium spp
4. ......................... staining is used in identification of Mycobacterial infection
5. ........................., ......................... & ......................... are examples of Gram Positive Bacteria
6. ........................., ......................... & ......................... are examples of Gram Negative Bacteria (ii) Motility testing Motility testing is performed by preparing a wet mount and is then observed under the microscope. Motility of bacteria can also be tested by inoculating the bacteria in the semisolid motility medium. (iii) Biochemical tests The staining is followed by use of various biochemical reagents and tests to get closer to the identification of bacteria. There are many biochemical

tests available for bacterial identification. Few of them are required to be carried out depending upon the bacteria. The commonly used biochemical tests are as mentioned below (a) Catalase test (b) Coagulase test (c) Oxidase test (d) Sugar fermentation test (e) Indole test (f) Citrate test (g) Urease test (a) Catalase test Purpose The catalase test facilitates the detection of the enzyme catalase in bacteria. It is essential for differentiating catalase-positive Micrococcaceae from catalasenegative Streptococcaceae. While it is primarily useful in differentiating between genera, it is also valuable in speciation of certain gram positives such as Aerococcus urinae (positive) from Aerococcus viridians (negative) and gramnegative organisms such as Campylobacter fetus , Campylobacter jejuni , and Campylobacter coli (all positive) from other Campylobacter species. Procedure: Place a microscope slide inside a petri dish. Keep the petri dish cover available. Using a sterile inoculating loop or wooden applicator stick, collect a small amount of organism from a well-isolated 18- to 24-hour colony and place it onto the microscope slide. Be careful not to pick up any agar. This is particularly important if the colony isolate was grown on agar containing red blood cells. Carryover of red blood cells into the test may result in a false-positive reaction. Using a dropper or Pasteur pipette, place 1 drop of 3% H 2 O 2 onto the organism on the microscope slide. Do not mix. Immediately cover the petri dish with a lid to limit aerosols and observe for immediate bubble formation (O 2 + water =