Isolation
Screening
Serial Dilution
Bacteria
Fungi
Algae
Protozoa
Amoeba
109 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10 METHODS OF ISOLATION OF BACTERIA 10.1 INTRODUCTION We have learned in earlier chapters that there exist so many bacteria that cause human disease.so now our task is to isolate these bacteria and identify them. The identification is required so as to cure the illness or the infection caused due to these bacteria, using appropriate antibiotics. Identification also holds significance for epidemiological purposes. This chapter would focus on various methods used for isolation of bacteria. While in subsequent chapters we would learn about identification of bacteria and the ways to contain the infections caused by them. OBJECTIVES After reading this chapter, you will be able to : z Expalin the steps involved in the isolation of bacteria. z describe the significance of Specimen collection. z describe the significance of Preservation and transportation of specimen. z explain the role of microscopy in isolation of bacteria. z explain various methods for isolation of bacteria. 10.2 ISOLATION OF BACTERIA Isolation of bacteria forms a very significant step in the diagnosis and management of the illness. Isolation of bacteria involves various steps – z Specimen collection z Preservation and transportation of specimen MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 110 Notes z Microscopic examination of sample z Various methods used for isolation of bacteria Specimen collection Many different specimens are sent for microbiological examination from patients with suspected bacterial infection. Common specimens include urine, faeces, wound swabs, throat swabs, vaginal swabs, sputum, and blood. Less common, but important specimens include cerebrospinal fluid, pleural fluid, joint aspirates, tissue, bone and prosthetic material (e.g. line tips). 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. 10.1: 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. 111 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 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. 10.2: 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 specimens before they reach the laboratory. In particular urine and other specimens that utilize a semiquantitative culture technique for thier detection, as delays in transport can give rise to falsely high bacterial counts when the specimen is processed. Microscopy A Gram stain helps with the visualization of bacteria, and gives an indication of the type of bacteria present, based on the shape of the bacteria and the staining MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 112 Notes properties (Gram positive: purple; Gram negative: pink/red). A Gram stain also helps to identify mixtures of bacteria, helps to determine the appropriate range of agar plates to be used for subsequent culture, and helps with the interpretation of culture results. Fig. 10.3: Gram positive cocci Fig. 10.4: Gram negative bacilli For liquid specimens e.g. CSF, the sample is first centrifuged to concentrate any bacterial cells in the deposit, and Gram stain and culture 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 10.1 1. Specimens that contain pathogens which are hazardous to laboratory staff should be labeled as .................. 2. Swabs are sent to laboratory in .................. medium 113 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 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 10.3 METHODS OF ISOLATION OF BACTERIA Methods of isolation of bacteria can be broadly classified into two z Culture methods z On Solid media z On Liquid media z Automated systems z Non-culture methods Culture methods 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 z Optimal atmospheric conditions z Optimal temperature z 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. MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 114 Notes Fig. 10.5: Incubator Growth requirement of the bacteria Different bacteria have different growth requirements. For eg Streptococcus pneumoniae requires factor V and factor X for its growth, which are found in chocolate agar. Thus for sample suspected of S. pneumoniae the samples are plated on chocolate agar. Similarly depending upon the growth requirements the appropriate culture media are used. INTEXT QUESTIONS 10.2 1. .................. & .................. methods are commonly used methods for bacterial isolation 2. Colonies of bacteria can be identified after .................. hours of incubation 3. The optimum temperature most bacteria require to grow are .................. 4. Chocolate agar has .................. & .................. which is used in the diagnosis of streptococci Pneumonia 115 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10.4 CULTURE ON SOLID MEDIA The principal method for the detection of bacteria from clinical specimens is by culture on solid culture media. Bacteria grow on the surface of culture media to produce distinct colonies. 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. 10.6: Streaking method MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 116 Notes 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 from the 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 then spread 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 >105Colony forming units per millilitre of urine. The method of inoculating the solid culture media is as shown in the figure. Fig. 10.7: Inoculation methods Fig. 10.8: Uninoculated Mac conkey Agar and Blood agar plate 117 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes Fig. 10.9: Lactose fermenting (pink coloured) colonies on mac conkey agar Culture in liquid media Bacteria can also be grown in liquid media (broth). Like agar plates, broth cultures may be non selective or selective. Bacterial growth is easy to detect as the clear liquid turns turbid, usually within 24–48 hr, but incubation may need to be extended to 14 days or more. The advantage of broth culture is that it is significantly more sensitive than direct culture on agar. The disadvantage is that, by itself, it is not easy to determine the type of bacteria present or whether a mixed growth has occurred, and in most cases the broth must be subcultured onto solid agar plates. This causes an additional delay in culture results. Broth cultures are also prone to contamination. Broth enrichment media are used when high sensitivity is required e.g. for detection of bacteria from CSF, or to detect small numbers of Salmonella in a stool sample containing many millions of other bacteria. Fig. 10.10: 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 CO2 production). MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 118 Notes Fig. 10.11: Bactec Fig. 10.12: 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. 119 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes The following are some of the factors that are considered in interpreting bacteriological culture results: z type of specimen z any delays in processing z types of bacteria recovered z knowledge of the normal human flora at different sites z clinical information provided on the request form z details of recent antibiotic therapy There must be good liaison between healthcare workers and the microbiology laboratory, in order to ensure that the most appropriate investigations are performed, results are interpreted correctly, and clinically relevant bacteriological reports are produced. INTEXT QUESTIONS 10.3 1. .................... is used as gelling agar in culture media 2. .................... culture media grow a wide variety of bacteria 3. .................... is an example of selective media 4. For obtaining the isolated colonies .................... method is common method of inoculating 5. .................... is the liquid medium in which bacteria may be grown 6. Examples of Amplication techniques are ...................., .................... & .................... WHAT YOU HAVE LEARNT z Isolation of bacteria forms a very significant step in the diagnosis and management of the illness. Isolation of bacteria involves various steps – Specimen collection, Preservation and transportation of specimen, Microscopic examination of sample.Various methods used for isolation of bacteria culture methods which includes culture on solid or liquid media and automated system. Non culture methods include the molecular techniques eg PCR, SDA, NASBA. MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 120 Notes TERMINAL 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 culture media 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 ANSWERS TO INTEXT QUESTIONS 10.1 1. High-risk 2. Charcoal 3. 4oC 4. Gram stain 5. Ziehl-Neelson 6. Dark Ground 10.2 1. Direct culture & Non-culture 2. 18-24 3. 37oC 4. Factor V & Factor X 121 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10.3 1. Agar 2. Non-selective 3. MacConkey 4. Streaking 5. Broth 6. Polymerase Chain Reaction, Ligase Chain Reaction, Nucleic Acid Sequence Based Amplification
Screening
Serial Dilution
Bacteria
Fungi
Algae
Protozoa
Amoeba
109 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10 METHODS OF ISOLATION OF BACTERIA 10.1 INTRODUCTION We have learned in earlier chapters that there exist so many bacteria that cause human disease.so now our task is to isolate these bacteria and identify them. The identification is required so as to cure the illness or the infection caused due to these bacteria, using appropriate antibiotics. Identification also holds significance for epidemiological purposes. This chapter would focus on various methods used for isolation of bacteria. While in subsequent chapters we would learn about identification of bacteria and the ways to contain the infections caused by them. OBJECTIVES After reading this chapter, you will be able to : z Expalin the steps involved in the isolation of bacteria. z describe the significance of Specimen collection. z describe the significance of Preservation and transportation of specimen. z explain the role of microscopy in isolation of bacteria. z explain various methods for isolation of bacteria. 10.2 ISOLATION OF BACTERIA Isolation of bacteria forms a very significant step in the diagnosis and management of the illness. Isolation of bacteria involves various steps – z Specimen collection z Preservation and transportation of specimen MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 110 Notes z Microscopic examination of sample z Various methods used for isolation of bacteria Specimen collection Many different specimens are sent for microbiological examination from patients with suspected bacterial infection. Common specimens include urine, faeces, wound swabs, throat swabs, vaginal swabs, sputum, and blood. Less common, but important specimens include cerebrospinal fluid, pleural fluid, joint aspirates, tissue, bone and prosthetic material (e.g. line tips). 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. 10.1: 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. 111 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 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. 10.2: 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 specimens before they reach the laboratory. In particular urine and other specimens that utilize a semiquantitative culture technique for thier detection, as delays in transport can give rise to falsely high bacterial counts when the specimen is processed. Microscopy A Gram stain helps with the visualization of bacteria, and gives an indication of the type of bacteria present, based on the shape of the bacteria and the staining MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 112 Notes properties (Gram positive: purple; Gram negative: pink/red). A Gram stain also helps to identify mixtures of bacteria, helps to determine the appropriate range of agar plates to be used for subsequent culture, and helps with the interpretation of culture results. Fig. 10.3: Gram positive cocci Fig. 10.4: Gram negative bacilli For liquid specimens e.g. CSF, the sample is first centrifuged to concentrate any bacterial cells in the deposit, and Gram stain and culture 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 10.1 1. Specimens that contain pathogens which are hazardous to laboratory staff should be labeled as .................. 2. Swabs are sent to laboratory in .................. medium 113 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 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 10.3 METHODS OF ISOLATION OF BACTERIA Methods of isolation of bacteria can be broadly classified into two z Culture methods z On Solid media z On Liquid media z Automated systems z Non-culture methods Culture methods 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 z Optimal atmospheric conditions z Optimal temperature z 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. MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 114 Notes Fig. 10.5: Incubator Growth requirement of the bacteria Different bacteria have different growth requirements. For eg Streptococcus pneumoniae requires factor V and factor X for its growth, which are found in chocolate agar. Thus for sample suspected of S. pneumoniae the samples are plated on chocolate agar. Similarly depending upon the growth requirements the appropriate culture media are used. INTEXT QUESTIONS 10.2 1. .................. & .................. methods are commonly used methods for bacterial isolation 2. Colonies of bacteria can be identified after .................. hours of incubation 3. The optimum temperature most bacteria require to grow are .................. 4. Chocolate agar has .................. & .................. which is used in the diagnosis of streptococci Pneumonia 115 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10.4 CULTURE ON SOLID MEDIA The principal method for the detection of bacteria from clinical specimens is by culture on solid culture media. Bacteria grow on the surface of culture media to produce distinct colonies. 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. 10.6: Streaking method MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 116 Notes 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 from the 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 then spread 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 >105Colony forming units per millilitre of urine. The method of inoculating the solid culture media is as shown in the figure. Fig. 10.7: Inoculation methods Fig. 10.8: Uninoculated Mac conkey Agar and Blood agar plate 117 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes Fig. 10.9: Lactose fermenting (pink coloured) colonies on mac conkey agar Culture in liquid media Bacteria can also be grown in liquid media (broth). Like agar plates, broth cultures may be non selective or selective. Bacterial growth is easy to detect as the clear liquid turns turbid, usually within 24–48 hr, but incubation may need to be extended to 14 days or more. The advantage of broth culture is that it is significantly more sensitive than direct culture on agar. The disadvantage is that, by itself, it is not easy to determine the type of bacteria present or whether a mixed growth has occurred, and in most cases the broth must be subcultured onto solid agar plates. This causes an additional delay in culture results. Broth cultures are also prone to contamination. Broth enrichment media are used when high sensitivity is required e.g. for detection of bacteria from CSF, or to detect small numbers of Salmonella in a stool sample containing many millions of other bacteria. Fig. 10.10: 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 CO2 production). MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 118 Notes Fig. 10.11: Bactec Fig. 10.12: 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. 119 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes The following are some of the factors that are considered in interpreting bacteriological culture results: z type of specimen z any delays in processing z types of bacteria recovered z knowledge of the normal human flora at different sites z clinical information provided on the request form z details of recent antibiotic therapy There must be good liaison between healthcare workers and the microbiology laboratory, in order to ensure that the most appropriate investigations are performed, results are interpreted correctly, and clinically relevant bacteriological reports are produced. INTEXT QUESTIONS 10.3 1. .................... is used as gelling agar in culture media 2. .................... culture media grow a wide variety of bacteria 3. .................... is an example of selective media 4. For obtaining the isolated colonies .................... method is common method of inoculating 5. .................... is the liquid medium in which bacteria may be grown 6. Examples of Amplication techniques are ...................., .................... & .................... WHAT YOU HAVE LEARNT z Isolation of bacteria forms a very significant step in the diagnosis and management of the illness. Isolation of bacteria involves various steps – Specimen collection, Preservation and transportation of specimen, Microscopic examination of sample.Various methods used for isolation of bacteria culture methods which includes culture on solid or liquid media and automated system. Non culture methods include the molecular techniques eg PCR, SDA, NASBA. MICROBIOLOGY MODULE Methods of Isolation of Bacteria Microbiology 120 Notes TERMINAL 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 culture media 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 ANSWERS TO INTEXT QUESTIONS 10.1 1. High-risk 2. Charcoal 3. 4oC 4. Gram stain 5. Ziehl-Neelson 6. Dark Ground 10.2 1. Direct culture & Non-culture 2. 18-24 3. 37oC 4. Factor V & Factor X 121 Methods of Isolation of Bacteria MICROBIOLOGY MODULE Microbiology Notes 10.3 1. Agar 2. Non-selective 3. MacConkey 4. Streaking 5. Broth 6. Polymerase Chain Reaction, Ligase Chain Reaction, Nucleic Acid Sequence Based Amplification
Obtaining Pure Culture of Microorganisms: 6 Methods
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The following points highlight the top six methods used for obtaining pure culture of microorganisms. The methods are: 1. Streak Plate Method 2. Pour Plate Method 3. Spread Plate Method 4. Serial Dilution Method 5. Single Cell Isolation Methods 6. Enrichment Culture Method.
1. Streak Plate Method:
This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium (Fig. 16.13). The successive streaks “thin out” the inoculum sufficiently and the micro-organisms are separated from each other.
It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. These plates are incubated to allow the growth of colonies. The key principle of this method is that, by streaking, a dilution gradient is established across the face of the Petri plate as bacterial cells are deposited on the agar surface.
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Because of this dilution gradient, confluent growth does not take place on that part of the medium where few bacterial cells are deposited. Presumably, each colony is the progeny of a single microbial cell thus representing a clone of pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/needle and re-streaked onto fresh media to ensure purity.
2. Pour Plate Method:
This method involves plating of diluted samples mixed with melted agar medium (Fig. 16.14). The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium.
Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The bacteria and the melted medium are mixed well.
The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.
Pour plate method has certain disadvantages as follows:
(i) The picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and
(ii) The microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms.
However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.
3. Spread Plate Method:
In this method (Fig. 16.15), the mixed culture or microorganisms is not diluted in the melted agar medium (unlike the pour plate method); it is rather diluted in a series of tubes containing sterile liquid, usually, water or physiological saline.
A drop of so diluted liquid from each tube is placed on the center of an agar plate and spread evenly over the surface by means of a sterilized bent-glass-rod. The medium is now incubated.
When the colonies develop on the agar medium plates, it is found that there are some plates in which well-isolated colonies grow. This happens as a result of separation of individual microorganisms by spreading over the drop of diluted liquid on the medium of the plate.
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The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In contrast to pour plate method, only surface colonies develop in this method and the microorganisms are not required to withstand the temperature of the melted agar medium.
4. Serial Dilution Method:
As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media.
A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions. The inoculum is subjected to serial dilution in a sterile liquid medium, and a large number of tubes of sterile liquid medium are inoculated with aliquots of each successive dilution.
The aim of this dilution is to inoculate a series of tubes with a microbial suspension so dilute that there are some tubes showing growth of only one individual microbe. For convenience, suppose we have a culture containing 10 ml of liquid medium, containing 1,000 microorganisms (Fig. 16.16.), i.e., 100 microorganisms/ml of the liquid medium.
If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid medium, we would then have 100 microorganisms in 10 ml or 10 microorganisms/ml. If we add 1 ml of this suspension to another 9 ml. of fresh sterile liquid medium, each ml would now contain a single microorganism.
If this tube shows any microbial growth, there is a very high probability that this growth has resulted from the introduction of a single microorganism in the medium and represents the pure culture of that microorganism.
5. Single Cell Isolation Methods:
An individual cell of the required kind is picked out by this method from the mixed culture and is permitted to grow.
The following two methods are in use:
i. Capillary pipette method:
Several small drops of a suitably diluted culture medium are put on a sterile glass-coverslip by a sterile pipette drawn to a capillary. One then examines each drop under the microscope until one finds such a drop, which contains only one microorganism. This drop is removed with a sterile capillars pipette to fresh medium. The individual microorganism present in the drop starts multiplying to yield a pure culture (Fig. 16.17).
ii. Micromanipulator method:
Micromanipulators have been built, which permit one to pick out a single cell from a mixed culture. This instrument is used in conjunction with a microscope to pick a single cell (particularly bacterial cell) from a hanging drop preparation. The micro-manipulator has micrometer adjustments by means of which its micropipette can be moved right and left, forward, and backward, and up and down.
A series of hanging drops of a diluted culture are placed on a special sterile coverslip by a micropipette. Now a hanging drop is searched, which contains only a single microorganism cell.
This cell is drawn into the micropipette by gentle suction and then transferred to a large drop of sterile medium on another sterile coverslip. When the number of cells increases in that drop as a result of multiplication, the drop is transferred to a culture tube having suitable medium. This yields a pure culture of the required microorganism.
The advantages of this method are that one can be reasonably sure that the cultures come from a single cell and one can obtain strains with in the species. The disadvantages are that the equipment is expensive, its manipulation is very tedious, and it requires a skilled operator. This is the reason why this method is reserved for use in highly specialised studies.
6. Enrichment Culture Method:
Generally, it is used to isolate those microorganisms, which are present in relatively small numbers or that have slow growth rates compared to the other species present in the mixed culture.
The enrichment culture strategy provides a specially designed cultural environment by incorporating a specific nutrient in the medium and by modifying the physical conditions of the incubation. The medium of known composition and, specific condition of incubation favours the growth of desired microorganisms but, is unsuitable for the growth of other types of microorganisms.
A number of methods are available for the isolation of bacteria. The following are a few important methods:
1. Surface plating:
It is also called as streak culture method. The specimen to be cultured is taken in a platinum loop. One lapful of the specimen is transferred on to the surface of a well dried plate. Then it is spread over a small area at the periphery. The inoculum is then distributed thinly over the plate. This is done by streaking with the loop in a series of parallel lines in different segments of the plate. The loop should be flamed and cooled between different sets of streaks. After incubating the plate, it can be seen that growth is confluent at the original site of inoculation. But it becomes progressively thinner. In the final series of streaks, well separated individual colonies of bacteria can be obtained.
2. Enrichment and selective media:
Bacteria can be isolated by growing in enrichment or selective media. In these media, substances which inhibit the growth of unwanted bacteria is added. So there is a growth of only the bacteria which is wanted.
3. Aerobic and anaerobic conditions:
Aerobic and anaerobic bacteria can be separated by cultivation under aerobic or anaerobic conditions. The most widely used medium for anaerobic bacteria is Robertson's cooked meat medium. It contains fat-free minced cooked meat in broth. It is covered with a layer of sterile Vaseline.
4. Isolation by difference in temperature:
Thermophile bacteria grow at 60° C. Some bacteria like N. magnitudes grow at 22° C. By incubation at different temperatures, bacteria can be selectively isolated.
5. Separation of vegetative and spore forming bacteria:
Vegetative bacteria are killed at 80° C. But spore forming bacteria like tetanus bacilli survive at this temperature. So by heating at 80° C vegetative bacteria can be eliminated and spore forming bacteria can be isolated.
6. Separation of motile and non-motile bacteria:
This can be achieved by using Craig's tube or a U tube. In the U tube, the organisms are introduced in one limb and the motile organism can be isolated at the other limb.
7. Animal inoculation:
Pathogenic bacteria can be isolated by inoculation into appropriate animals, e.g. Anthrax bacilli can be isolated by inoculation into mice or guinea pigs.
8. Filtration:
Bacteria of different sizes may be separated by using selective filters.
9. Micromanipulation:
By means of micromanipulation, single bacterium can be separated and cultured.