Anaerobic culture methods skill based learning

Anaerobic culture methods skill based learning

1 MLT 2324 Medical Microbiology 3 Course Outline Subject Name : Medical Microbiology 3 Subject Code : MLT2324 Contact Hour/Week : Theory & tutorial 3 hour Lab 2 hour Assessment :

Final examination : 60 % Mid term : 20% Quiz : 5% Attendance & Participitation : 5 % Learning Objective 3 Know laboratory techniques concerning bacteria pathogens Perform serological test Explain nosocomial infection Explain the quality control and assurance in microbiology lab

Topics No Topic 1 Anaerobic techniques 2 Anaerobic bacteria 3 Respiratory infection 4 Gastrointestinal infection

5 Excretory system infection 6 Central nervous infection 7 Urinary system infection 8 Sexually transmitted disease 9 Nosocomial infection MID Term

Topics No Topic 10 Cut and pus 11 Mycological diagnostic Laboratory 12 Safety in virology laboratory 13 Collection and transport of viral sample

14 Virology diagnostic laboratory 15 Serological test ASO/ANA/IPP 16 Emerging and re-emerging pathogens infections Quality control programs 17 Final Lets the

learnin g Start 7 ANAEROBIC BACTERIA BASIC CULTURE METHODS Norazli Ghadin Learning Objectives 8

To describe what is anaerobic bacteria To discuss what is requirement for anaerobic bacteria culture To list appropriate samples for anaerobic bacteria examination To know the correct technique for anaerobic bacteria handling To know how to interpret the anaerobic bacteria examination What Are Anaerobic Microorganisms 9 Anaerobic microorganisms are widespread and very important

Do not require oxygen for growth - often extremely toxic Defining Anaerobes 10 Facultative anaerobes - can grow in the presence or absence of oxygen Obtain energy by both respiration and fermentation

Oxygen not toxic, some use nitrate (NO3-) or sulphate (SO42-) as a terminal electron acceptor under anaerobic conditions Strict Anaerobic Bacteria 11 Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor.

Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc. Culturing of anaerobes need special skills 12 Culture of anaerobes is extremely difficult because

need to exclude oxygen, slow growth and complex growth requirements By molecular methods based on DNA analysis and direct microscopy have shown that anaerobic bacteria are diverse The accepted specimens for anaerobic processing are as follows: 13 Sites CNS

Acceptable specimen CSF, abscess, tissue Dental/ ENT Abscess, aspirates, tissues

The accepted specimens for anaerobic processing are as follows: 14 Local abscess Trans tracheal aspirates, lung aspirates, pleural fluid, tissue, Protected bronchial washing

Pulmonary Needle aspirates The accepted specimens for anaerobic processing are as follows 15 Abdominal Urinary tract Genital tract

Ulcers/wounds Others Abdominal Abscess aspirate, fluid and tissues Suprapubic bladder aspirate Culdocentesis specimen, endometrial swabs

Aspirate/swab pus from deep pockets or from under skin flaps that have been decontaminated Deep tissue or bone lesions, blood, bone marrow, synovial fluid, tissues Interpretation by Physicians and Microbiologists 16

The physician who collected the specimen can best evaluate the anaerobic culture result. Interpretation of the result should be correlated with the clinical findings and how the specimen was collected. Clinical signs suggesting possible infection with anaerobes include the following: 1. Foul smelling discharge 2. Infection in proximity to a mucosal surface 3. Gas in tissues 4. Negative aerobic cultures of specimens

pus cells. Testing for anaerobes in Routine Practice 17 Deep culture tubes can be used to test whether an unknown organism is anaerobic/facultative or aerobic Thiglyclolate added to culture medium, oxygen only found near top where it can diffuse

from air -pattern of colony formation characteristic of organisms 18 Why Needle Aspiration Preferred for Anaerobic Bacteria II. Collection by needle aspiration is preferable than swab culture because of a. better survival of pathogen b. greater quantity of specimen c. less contamination

with extraneous organism are often achieved B. HANDLING 19 If a swab must be used, a 2 tube system must be used 1st tube contains swab in O2 free CO2 2nd tube contains PRAS (pre-reduced anaerobically sterilized culture media) Specimen should be placed in anaerobic transport device with gas mixture HANDING AND TRANSPORT OF CLINICAL SPECIMENS 20

The basic principles to remember are avoid contamination with the normal microbial flora prompt transport to the laboratory immediate processing is done. Transporting 21 Anaerobic transport tubes and/or devices

should always be available at the OR and ER. Specimens should be placed in leak-proof container with tight fitting caps. proper label for identification with date and time of collection should accompany all specimens submitted for culture. Put samples in room temperature while waiting for delivery to the laboratory. Some anaerobes are killed by refrigeration. 22 Anaerobic culturing Needs Define Chemicals and Environment

Pyrogallic acid-sodium hydroxide method can be used, again relies on a chemical reaction to generate an anaerobic environment, but a catalyst rather than a reducing agent Anaerobic jars (GasPak System) are sued to incubate plates in an anaerobic atmosphere, useful if brief exposure to oxygen is not lethal Anaerobic Culture Methods 23

Production of a vacuum Displacement of Oxygen with other gases Absorption of Oxygen by chemical or biological methods By using reducing agents P. aeruginosa Strict aerobe Enterococcus Facultative Grows aerobic or anaerobic. 24

Bacteriodes fragilis Obligate Anaerobes needs Optimal Methods 25 Obligate anaerobes can be culture in special reducing media such as sodium Thiglyclolate or in anaerobe chambers and handled in anaerobe

Displacement of Oxygen 26 By inert gases like Hydrogen, Nitrogen, Carbon dioxide or Helium Use of lighted candle - Use up Oxygen, but some Oxygen is left behind Vacuum decicator Unsatisfactory McIntosh & Fildes Jar 27

Hydrogen gas is passed in Catalyst helps to combine Hydrogen & O2 Reduced Methylene blue remains colorless if anaerobiosis is achieved Absorption of O2 by Chemical method 28 Pyrogallol Chromium

and sulphuric acid Gas-pak -available commercially By reducing agents 29 Thiglyclolate broth Robertsons Cooked Meat (RCM) broth contains nutrient broth with pieces of fat-free minced cooked meat of ox heart.

McIntosh & Fildes anaerobic Jar 30 Stout glass or metal jar with a lid Lid has an inlet for gas,outlet&2 terminals Alumina pellets coated with palladium (catalyst)

- under the lid Inoculated plates kept inside the jar Lid is clamped tight Air is evacuated A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture System 31 A. ANAEROBIC JAR 1. Candle Jar - reduces O2 environment - only CO2 tension 2. Gas Pak Jar a. Palladium aluminum coated pellets - catalyst

- chemically reduces O2 - reacts with residual O2 in the presence of H2 to form Culture of strict anaerobes 32 For culture of strict anaerobes all traces of oxygen must be removed from medium and for many organisms sample must be kept entirely anaerobic during manipulations Methanogenic archaea from rumen and sewage treatment plants killed by even a brief exposure to O2

Medium usually boiled during preparation and reducing agent added, stored under O2- Choosing the Optimal Media 33 Broth and solid media should both be inoculated. The culture media should include anaerobic blood agar plates enriched with substances such as brain-heart infusion, yeast extract, amino acids, and vitamin K; a selective medium such as kanamycinvancomycin (KV) blood agar or laked blood agar; and a broth such as brain heart infusion broth with Thiglyclolate or other reducing agent.

34 Media chosen according to our needs The choice of media depends upon the type of specimen. Some commonly used media include prereduced peptone-yeast extractglucose broth which is suitable for analysis of volatile products by gas chromatography; egg yolk agar for detection of lecithinase activity of Clostridium spp.; cycloserine-cefoxitin-fructose agar (CCFA) for isolation of Clostridium difficile from stool; and Bacteroides bile esculin agar for isolation of the Bacteroides fragilis group. A skilled plating the Medium is highly essential 35

Figure 6.10ab Anaerobic Glove Chamber 36 b. Gas Pak envelope - generates CO2 & H2 gases c. Methylene blue strip - indicator blue (+) O2 white (-) O2 II. Anaerobic Glove Chamber - close system - used for premature babies - e.g. incubator III. Roll Tube

- has a pedal gas ( CO2 & H2 ) would come out - place test tube directly to the outlet IDENTIFICATION of ANAEROBES 37 Plates are checked at > 18-24 hours for faster growing species like Cl. Perfringens & B.fragilis & daily thereafter up to > 5-7 days for slowly growing species like Actinomyces, Eubacterium & Propionibacterium Genus is determined by - gram stain, cellular morphology, Gas-liquid chromatography Species determination is based on fermentation of sugars & other biochemical determination

Identification of Anaerobes is Complex 38 The identification of anaerobes is highly complex, and laboratories may use different identification systems. Partial identification is often the goal. For example, there are six species of the Bactericides genus that may be identified as the Bactericides fragilis group rather than identified individually. Organisms are identified by their colonial and microscopic morphology, growth on selective media, oxygen tolerance, and All isolates to the Purified by Sub culturing

39 Isolated organisms are always subcultured and the pure culture is tested in order to identify the organism. The identification of anaerobes is highly complex, and laboratories may use different identification systems. Partial identification is often the goal. For example, there are six species of the Bacteroides genus that may be identified as the Bacteroides fragilis group rather than identified individually. Organisms are identified by their colonial and microscopic examination. Needs several Biochemical Tests for Identification 40

Organisms are identified by their colonial and microscopic morphology, growth on selective media, oxygen tolerance, and biochemical characteristics. These include sugar fermentation, bile solubility, esculin, starch, and gelatin hydrolysis, casein and gelatin digestion, catalase, lipase, lecithinase, and indole production, nitrate reduction, volatile fatty acids as determined by gas chromatography, and susceptibility to antibiotics. The antibiotic susceptibility profile is determined by the micro tube broth dilution method. Many species of anaerobes are Antibiotic Sensitivity Testing 41

.The antibiotic susceptibility profile is determined by the micro tube broth dilution method. Many species of anaerobes are resistant to penicillin, and some are resistant to clindamycin and other commonly used antibiotics

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