Chapter 16 - Introduction to Medical Microbiology PDF

Chapter 16 INTRODUCTION TO MEDICAL MICROBIOLOGY 1  Learning Outcomes 1. Name the four (4) distinctive fields of study in microbiology. 2. Compare a prokaryotic cell versus a eukaryotic cell. 3. When given an example, identify the genus and species of a microorganism...

Chapter 16 INTRODUCTION TO MEDICAL MICROBIOLOGY 1  Learning Outcomes 1. Name the four (4) distinctive fields of study in microbiology. 2. Compare a prokaryotic cell versus a eukaryotic cell. 3. When given an example, identify the genus and species of a microorganism. 4. Define pathogen, nosocomial infection (HAI), and opportunistic pathogen. 5. List the different methods of disinfection and sterilization in the microbiology lab. 6. Explain the importance of collection requirements, transportation, and specimens for microbiological examination. 7. List the basic equipment and techniques used in microbiology. 8. Explain the principle of the Gram stain reaction for gram- positive and gram-negative bacteria. 9. List the different morphological characteristics of bacteria. 10.Explain the dilution streak method for isolation of bacterial colonies. 2  Learning Outcomes 11. Define primary culture, subculture, and pure culture. 12. Define the terms aerobic and anaerobic. 13. List the classification of common media used in the microbiology laboratory. 14. Identify the three (3) types of hemolysis used to classify certain bacteria. 15. Define cystitis and pyelonephritis. 16. Explain the method used to streak and isolation organisms in a urine culture. 17. Identify antimicrobial susceptibility tests from photomicrographs shown. 18. Explain the different areas of quality control performed in the microbiology laboratory. 19. Identify the characteristics of fungi. 20. List the common parasites and their sources as discussed in the PowerPoint. 3  Introduction to Microorganisms https://youtu.be/9JW63U2mzqo? si=VvrktOCiCEn9B4aP The field of medical or clinical microbiology involves the isolation and identification of infectious organisms, and the development of effective ways to eliminate or control infectious organisms. Medical microbiology is generally divided into the study of bacteria, viruses, fungi, and parasites. The study of bacteria is bacteriology, the study of fungi is mycology, the study of parasites is parasitology, and the study of 4  Introduction to Microorganisms All living organisms can be classified according to their cellular makeup. Cells are defined as prokaryotic or eukaryotic. 5  Introduction to Microorganisms Prokaryotic and eukaryotic have cell differences Prokaryotes (Greek for “before nucleus”) do not have a nucleus (membrane-bound organelle containing chromosomes) or any other membrane-bound organelles such as mitochondria. For example: bacteria Eukaryotes (Greek for “true nucleus”) are organisms composed of eukaryotic cells and include animals, plants, and fungi. For example: parasites and fungi 6 Introduction to Microorganisms Fig. 15.1. Major features of prokaryotic and eukaryotic cells. (From Murray PR, Rosenthal KS, Pfaller MA: Medical microbiology, ed 5, Philadelphia, 2005, Mosby.) 7  Classification of Microorganisms: Taxonomy https://youtu.be/jet1l0g8unk?si=oiviGXzhlF_tcTXN The scientific study of and the classification process is known as taxonomy. Provides an orderly method for placing organisms into categories. Historically, categories were based on morphologic and biochemical properties. Now genetic testing has provided a more detailed and accurate method for classifying and grouping organisms. Taxonomy is also used as a standard method for developing a nomenclature of living things. Taxonomy provides a mechanism for the ID of living things and assigning them to a particular taxonomic gp, such as bacteria, protozoa, or fungi. 8 Classification of Microorganisms: Taxonomy With the use of biological classification methods, it is possible for the laboratory microbiologist to ID microbes systematically through the use of: A. Morphologic B. Biochemical C. Genetic characteristics The classification scheme is divided into the following categories: kingdom, phylum, class, order, family, genus, and species. There are five kingdoms within the classification scheme: Animalia, Plantae, Protista, Fungi, and Monera (also referred to as Prokaryotae). ◦ Bacteria belong to the kingdom Monera, parasites to the kingdom Protista, and yeasts and molds to the kingdom Fungi. 9 Classification of Microorganisms: Taxonomy Medically important microorganism include large groups of microorganism, and the family division may be used to include all members. The family division precedes the genus designation; in other words, similar genera are grouped into families. 10  Classification of Microorganisms: Taxonomy As part of the systematic classification, all organisms are designated with a minimum of binomial nomenclature (consisting of 2 names). Binomial nomenclature consists of the genus + species name. One genus may have many different species. ◦ EXAMPLE: Staphylococcus aureus, Staphylococcus epidermidis Normal flora ◦ Microorganisms are present under normal conditions in specific sites on or in the human body. ◦ In areas of the body with expected microbiota, host microbiota, and the immune system interact to maintain tissue homeostasis in healthy individuals. 11  Classification of Microorganisms: Taxonomy Normally sterile fluids and their common pathogens Blood ◦ Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Clostridium spp. Cerebrospinal fluid ◦ Neisseria meningitidis Urine ◦ E. coli, Proteus sp., Pseudomonas sp. Sputum ◦ Streptococcus pneumoniae, Staphylococcus aureus Stomach ◦ Helicobacter pylori 12  Classification of Microorganisms: Taxonomy Anatomic body locations, normal flora, pathogens Skin ◦ Normal flora: Staphylococcus sp. coagulase negative ◦ Pathogen: Staphylococcus aureus Intestines ◦ Normal flora: Escherichia coli, Pseudomonas sp. ◦ Pathogen: Salmonella sp., Shigella sp. Throat ◦ Normal flora: Alpha (α) streptococcus, commensal Neisseria sp. ◦ Pathogens: Group A-β hemolytic Streptococcus pyogenes Genitourinary ◦ Normal flora: None ◦ Pathogens: Neisseria gonorrhoeae, Chlamydia trachomatis; In vaginitis-Gardnerella vaginalis, Trichomonas vaginalis or Candida albicans 13  Classification of Microorganisms: Taxonomy Pathogenic microorganisms ◦ Microorganisms that cause disease are pathogens. ◦ The ability to cause disease is pathogenicity. ◦ Healthcare-associated infections (HAIs) or nosocomial infections are infections that patients acquire during treatment in a healthcare facility such as MRSA. ◦ An opportunistic pathogen does not usually cause an infectious disease in a person with an intact immune system but can cause disease when the host’s immune system has been compromised. ◦ https://youtu.be/JYRkXhT1XEs?si=nPHntpBgUaKE--8u 14 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization Classification of biological agents based on hazard to personnel ◦ Biosafety Level 1: minimal hazard (non-pathogenic strain ecoli) ◦ Biosafety Level 2: moderate threat (Salmonella, Shigella) ◦ Biosafety Level 3: not common in the laboratory (Yellow fever, TB) ◦ Require personnel to have specialized training and use special barrier devices ◦ Biosafety Level 4: highest risk (EBOLA) ◦ Not found in routine laboratories, pose high risk of aerosol transmission, are frequently fatal, and have no treatment or vaccine. ◦ CDC ◦ https://youtu.be/xNYASNEdgg8?si=F9LmgGAS_qVY6_5H 15 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization General safety practices in the microbiology laboratory ◦ Waste disposal process- ◦ use biohazardous bags/containers for culture media and patient specimens ◦ use puncture-resistant sharps containers for needles ◦ The decontamination process is completed by autoclaving, incineration, or burning. ◦ Disinfection process- disinfect lab benchtops before and after using 10% bleach 16 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization Incinerators and Flame Burners: Are used to sterilize inoculating loops and needles These units are electrical, as opposed to flame burners that operate on gas, and may still be used in research or teaching labs. Now disposable loops are used 17 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization Disinfection and Sterilization Techniques- Essential to use sterile media for growing pure cultures of bacteria and to avoid contamination by microorganisms in nature (organisms in the air, on the hands, and lab equipment and supplies) Contaminated media must be disposed of in biohazard bags 18 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization Types of Disinfection Techniques include chemical Disinfectants. A. Sterilization-killing spores of bacteria by involving physical means such as heat or filtration and chemical means such as oxidation B. Disinfection: destroying bacteria by physical means (dry heat) or chemicals (bleach 10%, phenols, aldehydes, and alcohols) C. Antisepsis: includes iodine and alcohol to disinfect the skin before drawing blood or venipuncture. 19 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization TYPES OF STERILIZATION TECHNIQUES: A.USE OF HEAT OR BURNING BY 1. Sterilization by dry heat 2. Sterilization by moist heat- The most effective means of sterilization with moist heat involves steam under pressure, using a special device called an autoclave 20 Protection of Laboratory Personnel, Decontamination, Disinfection, and Sterilization The USE OF FILTRATION: 1. Filtration through thin membrane filters may be used as an alternate method of sterilization 2. Heat Sensitive solutions such as vaccines or antibiotic solutions can be sterilized by filtration 3. HEPA filters, filter air in biological safety cabinets ( isolation and operating rooms use these) are capable of removing 99% of microorganisms larger than 0.3 microns 21 Specimens for Microbiological Examination Specimen collection requirements for culture ◦ Documentation for all specimens sent to the microbiology laboratory should include the source. If available, the anatomic location of the wound is important and it is also useful to indicate what antibiotics the patient may have received. ◦ Identification of the causative agent is required to initiate effective treatment. ◦ For each specimen, personnel collecting the specimen must follow appropriate procedures and be aware of the types of pathogens present in each type of specimen. 22  Specimens for Microbiological Examination Specimen containers ◦ Specimens must be collected in sterile containers and must not be contaminated during transfer to or isolation in the laboratory. ◦ Disposable containers ◦ Disposable culture units ◦ Anaerobic/ Aerobic transport systems 23  Specimens for Microbiological Examination ◦ Transport to the laboratory ◦ Specimens should be delivered to the laboratory promptly. ◦ Although many organisms remain viable for long periods after collection, some are fastidious, requiring special growth conditions, including rapid inoculation into a suitable nutrient medium. ◦ Some organisms are so fragile that the appropriate collection device and nutrient media are supplied to the patient so that the specimen can be placed directly into the container. 24 Specimens for Microbiological Examination ◦ Handling and storing specimens in the laboratory ◦ Immediate culture of freshly collected specimens is not always practical. ◦ Storage is not appropriate for some microorganisms (certain meningitis-causing bacteria in CSF are Susceptible to low temps) ◦ Refrigeration –will prevent overgrowth of normal flora in the specimen ◦ Refrigeration is not appropriate for some microorganisms (anaerobic bacteria, CSF, genital cultures for Neisseria, stay at RT) ◦ Freezing ◦ Serum samples for serology testing can be stored in the freezer for 1 week before testing. ◦ Only when specimens are properly collected and handled are the final results of the culture or test valid. 25  Specimens for Microbiological Examination Types of microbiology specimens collected ◦ Blood ◦ Body fluids ◦ Cerebrospinal fluid ◦ Inner ear ◦ Respiratory ◦ Scrapings ◦ Stool ◦ Sputum ◦ Swabs of various anatomical sites ◦ Urine 26  Basic Equipment and Techniques Used in Microbiology Most microbiology laboratory work involves procedures to culture, characterize, and identify various microbes ◦ Culture of organisms present in the patient specimens ◦ Classification and identification of the isolated organisms ◦ Interpretation of organism susceptibility patterns to determine an appropriate antimicrobial agent Inoculating needles or loop-were reusable but are now disposable (leading to a decrease in contamination) 27 Basic Equipment and Techniques Used in Microbiology In microbiology, the main difference between solid and liquid media is that solid media contains a solidifying agent, while liquid media does not: Solid media Contains a solidifying agent like agar, gelatin, or silica gel. This allows for the growth and isolation of individual colonies of microorganisms on the surface of the media.Solid media is used to establish pure cultures, isolate microbes, and make agar slants and agar stabs. Liquid media Also known as nutrient broths, these media are commonly used for growing microorganisms. Liquid media can be prepared with a variety of nutrient components, such as peptone, yeast extract, inorganic salts, and distilled water. 28  Basic Equipment and Techniques Used in Microbiology Fig. 15.5. (A) Example of bacterial growth on a solid blood agar plate. (B) Turbidity produced by bacteria growing in thioglycollate liquid broth. (From A. Mahon CR, Lehman DC, Manuselis: Textbook of Diagnostic Microbiology, ed. 5, 2015.) 29  Basic Equipment and Techniques Used in Microbiology Culturing techniques ◦Dilution streak technique ◦ The first streak is continued across approximately one quarter of the plate (first quadrant). The plate is then shifted about a one-quarter turn and streaked again, beginning at the periphery, overlapping the previously inoculated area a few times, and continuing across the second quadrant. The plate is turned once again and streaked a third time, beginning at the periphery, drawing the loop through the second streak a few times, and continuing across the third quadrant. The fourth quadrant is streaked beginning at the periphery, drawing the loop through the third streak a few times, and continuing across the fourth quadrant. 30 Basic Equipment and Techniques Used in Microbiology Fig. 15.6. Dilution streak technique. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 31 Basic Equipment and Techniques Used in Microbiology Incubators: A microbiology incubator is a heated, insulated chamber that maintains the ideal conditions for growing and maintaining microbiological cultures: Temperature: The incubator maintains a specific temperature for the growth of cells, which can vary depending on the organism. For example, bacteria like E. coli and mammalian cells grow well at around 99°F (37°C), while budding yeast grows best at 86°F (30°C). Humidity: The incubator maintains the ideal humidity level for the growth of cells. Gas composition: The incubator maintains the ideal gas composition for the growth of cells, such as the carbon dioxide (CO2) and oxygen content. Sterility: The incubator maintains a sterile environment for the growth of cells. 32  Identification of Bacteria The identification of most bacteria involves microscopic observations (smear preparation and staining), bacterial cultivation, and biochemical tests. A. Smear preparation and stains used in microbiology: ◦ Smear preparation: to visualize the presence of bacteria, the specimen is spread thinly on a glass microscope slide and allowed to air dry. ◦ Staining procedures used (gram stain) yield valuable information like the examination of cultures to determine purity. GS on CSF and BFs were considered STAT results and had to be called (sterile fluids). ◦ Types of Stain: Differential stains use more than one stain to provide additional information about bacteria. Cells from different bacterial species can appear different based on their chemical or structural properties. ◦ GPC stains purple and GNR stains pink ◦ Simple stains- methylene blue stain - used in microbiology to stain cells, tissues, and other specimens to highlight their structures, shapes, and locations. MANY OTHERS! 33 34 Identification of Bacteria ◦ Involves morphologic examination under the microscope (BRIGHTFIELD MICROSCOPY) ◦ Morphology of bacteria: ◦ Spherical or round bacteria are cocci (singular coccus) ◦ The prefix diplo- describes bacteria that occur in pairs, strepto- describes bacteria occurring in chains, and staphylo- refers to irregular clumps or clusters of bacterial cells. ◦ Rod-shaped bacteria are bacilli (singular bacillus) ◦ Curved rods are called vibrios ◦ Spiral-shaped bacteria are spirochetes and spirilla (singular spirochete and spirillum). 35 Identification of Bacteria Fig. 15.9. Bacterial morphology. Shown are characteristic shapes and cellular arrangements of bacteria. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 36  Identification of Bacteria The bacterial cultivation process of identification of microorganisms begins by inoculating various nutrient media designated to enhance the growth of the pathogen bacteria. ◦Primary culture, subculture, and pure culture ◦ When culturing specimens, the primary culture plates are inoculated using the dilution streak technique to obtain isolated colonies. ◦ If the colonies on the primary plates appear as mixtures of more than one species of bacteria, a subculture (secondary streak plate) must be performed to separate the different types of bacteria. ◦ The growth of several colonies originating from a single colony, and thus a single cell, is known as a pure culture. 37 Identification of Bacteria T Y PE S O F C U LT U R E M E D IA - A G A R PLAT E S , A G A R S LA N T S , B R O T H S (LI Q U ID M E D IA ) Colony characteristics (appearance) of bacterial cultures: when inoculated and with proper temp and moisture bacteria rapidly multiply and form macroscopic colonies. 38 Identification of Bacteria Fig. 15.11. Pure culture of Staphylococcus aureus on a sheep blood agar plate. Each colony began as an individual parent bacterial cell that multiplied many times to become visible. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 39 IDENTIFICATION OF BACTERIA Requirements for bacterial cultivation involve specific requirements to sustain life and reproduce. The culture requirements for bacteria include a source of nutrients, proper temp, an adequate supply of oxygen, and the correct pH. For example, Oxygen requirements for Aerobes and anaerobes ◦ Aerobes = grow with oxygen ◦ Anaerobes = grow without oxygen ◦ Nutrients- different bacteria require various types of nutrients for energy production and metabolism. For example, some grow on media containing simple mixtures of inorganic salts. 40  Identification of Bacteria ◦ Temperature- bacteria grow at a wide range of temps (35C) ◦ pH (hydrogen ion concentration)- culture medium must contain proper nutrients in correct concentrations but must also have the correct degree of acidity or alkalinity. Most bacteria like media with a pH between 6.5 to 7.5 ◦ Sterile conditions- culture media must be sterile ◦ Moisture- necessary for metabolic reactions, dehydration= reduces bacterial growth. 41 Identification of Bacteria ◦Incubation time and temperature for routine cultures: ◦ Bacterial growth on artificial media or in culture typically requires 24 to 48 hours, although anaerobic cultures require longer incubation times, usually 3 to 5 days, to reach sufficient growth required for the identification of the bacteria. 42  Identification of Bacteria ◦Storage of media- under refrigeration 4C to prevent deterioration and dehydration 43 Identification of Bacteria ◦ Different types of media are used in micro to aid not only in supporting growth but also in identification. Therefore, media are placed in categories according to their specific use: ◦ Classification of media ◦ Enrichment media- one bacteria grows in the presence of specific nutrients ◦ Supportive media- water and nutrients that support the growth of microorganisms (THIO broth) ◦ Selective media –inhibit the growth of gram-positive bacteria ◦ Differential media- It differentiates between microorganisms based on their biochemical properties by incorporating specific chemicals or indicators that react differently depending on the organism's metabolism. (For example, MAC diff bacteria on the ability to ferment lactose, NLF, and F) 44  Identification of Bacteria- Selective Media- LF Ecoli, NLF pseudomonas Fig. 15.14. MacConkey agar (MAC). MAC is a selective and differential medium used frequently for primary plating of specimens to isolate and differentiate gram-negative bacilli. (A) Gram- negative bacilli that ferment lactose will appear pink/red. (B) Gram-negative bacilli that do not ferment lactose will appear clear or slightly pink. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 45  Identification of Bacteria ◦Various types of media: ◦Chocolate agar (Choc)- fastidious bacteria like Haemophilus and Neisseria ◦Hektoen enteric agar (HE)- Salmonella, Shigella ◦MacConkey agar (MAC)- GN bacteria ◦Sheep blood agar (BAP)- supports growth of most bacteria ◦Thayer-Martin agar (modified Thayer-Martin agar)-gonorrhea 46  Identification of Bacteria Fig. 15.15. Hektoen enteric (HE) agar. HE agar is a selective and differential medium for the isolation of enteric pathogens. (A) Escherichia coli is a lactose-fermenting gram-negative bacillus and appears yellow on HE. (B) Shigella spp. do not ferment lactose and will appear green or transparent. (C) Salmonella spp. are also non–lactose-fermenters but produce hydrogen sulfide (H 2S); therefore the colonies will appear green or transparent with black centers. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 47 Identification of Bacteria-Lysine Iron Agar LIA is used to distinguish bacteria that can produce hydrogen sulfide and decarboxylate lysine from those that cannot. It's especially useful for distinguishing Gram- negative bacilli, particularly those in the Enterobacteriaceae 48 Identification of Bacteria- BAP (blood agar plate) Fig. 15.17. Three characteristic types of hemolysis on sheep blood agar: (A) Alpha hemolysis of Streptococcus pneumoniae, (B) Beta hemolysis of Staphylococcus aureus, and (C) Gamma hemolysis of Enterococcus faecalis. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014.) 49 Quality Control of Media Quality control of media in microbiology is a process to ensure that the media used in a laboratory is of good quality and can produce accurate results. This is important because the quality of the media directly affects the observations and conclusions drawn from the microorganisms. 50 Identification of Bacteria Biochemical or enzymatic tests- Many bacteria can't be ID based on microscopic or culture characteristics alone. ◦ In multi-test systems, conventional biochemical tests are arranged in a series. ◦ Bile esculin agar- ◦ Aids in the identification of enterococci ◦ Bile solubility test- ◦ Aids in the identification of S. pneumoniae ◦ Catalase test- ◦ Often used to differentiate staphylococci from streptococci 51 Identification of Bacteria ◦ Citrate utilization ◦ Useful in the identification of members of the Enterobacteriaceae ◦ Coagulase test ◦ Used to differentiate S. aureus from other members of the genus Staphylococcus ◦ Indole test: Used to differentiate swarming Proteus species and as a presumptive identification of E. coli https://youtu.be/MYAl0mCSBxw?si=oTUVtNEA14_cCZMt 52  Identification of Bacteria An API test panel, or Analytical Profile Index test panel, is a set of biochemical test strips that identify bacteria and fungi: How it works: Each strip contains a series of cupules with freeze-dried reagents and color indicators. A sample containing the microbe is inoculated into the cupules, and the strips are incubated. The results generate a code that 53 can be used to identify the microbe in a database.  Urine Cultures Cultures are done on urine to diagnose bacterial infections of the urinary tract. Urinary tract infections (UTIs) are of two main types: ◦ Lower UTIs of the bladder or urethra, such as cystitis ◦ Upper UTIs of the ureters and kidneys, such as pyelonephritis 54  Urine Cultures Collecting the specimen ◦Interpretive guidelines for urine specimens ◦ Type of specimen-UV or UC ◦ Number of potential pathogens->100 CFU of ECOLI ◦ Number of colony-forming units per milliliter ◦ A complete workup includes identification of the organism and appropriate susceptibility testing ◦ Three or more organism types with none predominating from a clean-catch midstream specimen or catheterized specimen suggest contamination; ask for another specimen. 55  Urine Cultures Methods for detection of urinary tract infections ◦Rapid-screening test strips- Rapid- screening test strips, also known as urine dipsticks, are a common method for detecting urinary tract infections (UTIs) by analyzing a urine sample for the presence of substances like leukocytes (white blood cells) and nitrites, which are often elevated in individuals with a UTI 56 Urine Cultures Quantitative culture methods are used in clinical microbiology laboratories to determine the bacterial burden in a sample and whether it's clinically significant. The streak plate method is a microbiology technique that separates pure colonies of bacteria from a mixed population. It's based on the principle of dilution and is used to isolate individual cells so they can be counted as colony- forming units (CFUs). 57 Urine Specimens Gram stain of urine specimens (not routinely done): A Gram stain of urine is a test that helps identify bacteria in a urine sample to diagnose a bacterial infection: How it works A purple stain is used to color bacteria in a sample. The bacteria will either retain the purple color (Gram-positive) or turn pink (Gram-negative). What it shows The Gram stain groups bacteria by color and shape to help determine the type of bacteria causing an infection 58 59  Urine Cultures Fig. 15.21. Method for inserting a calibrated loop into urine to ensure that the proper amount of specimen will adhere to the loop. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 60 Urine Cultures Fig. 15.22. Method for streaking a loopful of urine with a calibrated loop to produce isolated colonies and countable colony-forming units of organisms. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 61  Throat Cultures Collecting the specimen Methods for detection of group A β- hemolytic streptococci ◦ Rapid detection methods: Non-culture techniques ◦ Culture on sheep blood agar- addition of bacitracin disc (Taxo A) ◦ Interpreting results of throat culture plates-colony morphology & hemolysis on BAP ◦ Appearance of hemolysis- alpha, beta, gamma ◦ Identification of group A β-hemolytic streptococci ◦ Molecular diagnostic testing for streptococci-PCR For detection of gp A or B (DNA) 62 Beta-hemolysis: Complete lysis of red blood cells around the colony Alpha-hemolysis: Partial lysis of red blood cells, also known as "greening" Gamma-hemolysis: No hemolysis 63  Blood Cultures Blood is normally sterile. 64 Blood Cultures- bacteria isolated from blood Staphylococci: Coagulase-negative staphylococci are the most frequently isolated bacteria from blood cultures. Staphylococcus aureus is also common. Enterococci: Enterococcus faecalis and Enterococcus faecium are common isolates. Escherichia coli: A common isolate. Klebsiella pneumoniae: A common isolate. Streptococcus pneumoniae: A common isolate. Pseudomonas aeruginosa: A common isolate. Enterobacter cloaca: A gram-negative rod that is often isolated. Salmonella typhi: A gram-negative rod that is often isolated. Acinetobacter baumannii: A gram-negative rod that is often isolated 65 Blood Cultures Collecting the specimen ◦ Cleansing the collection site- 70% alcohol and a povidone-iodine solution Culture media for blood- Here are some common types of blood culture media: Broth media: These media support the growth of bacteria and some fungi. Some examples include tryptic soy broth, brain heart infusion broth, and thioglycolate broth. Columbia broth: This broth is good for anaerobic bacteria. Pre-reduced peptone broths: These broths are good for anaerobic bacteria. Thio broths: These broths are good for anaerobic bacteria. 66  Examination of Bldc’s Blood cultures are laboratory tests that use Bactec media to detect the presence of bacteria, fungi, viruses, and other germs in a blood sample: Bactec media A range of media that can detect aerobes, anaerobes, yeast, fungi, and mycobacteria. Detection methods Different Bactec systems use different methods to detect growth, including pressure changes, color changes, and fluorescence. Radiometric method A method that measures the amount of 14CO2 produced by bacteria metabolizing 14C labeled palmitic acid in the culture's liquid media. Time to detection The BACTEC system can detect growth faster than other systems, such as the BacT/Alert system. Incubation time If no growth is detected, the blood culture bottles may be incubated for up to two weeks. 67 Automated System for Blood cultures 68 Antimicrobial Susceptibility Tests Antimicrobial susceptibility tests (ASTs) are medical tests that determine which antibiotics or other antimicrobial agents are most effective against a specific microorganism causing an infection. ASTs are performed by clinical microbiology laboratories and are used to help physicians select the appropriate treatment for a patient. ASTs are important because: Identify the specific diagnosis ASTs help identify the specific diagnosis and the causative agent of the disease. Target the appropriate treatment ASTs help ensure that patients receive the appropriate treatment, as the same causative organism can have different resistance patterns. Address antimicrobial resistance AST results are an important component in addressing the problem of antimicrobial resistance. There are several types of ASTs, including: Broth microdilution: A widely used testing method Rapid automated instrument methods: Use commercially marketed materials and devices Disk diffusion: A manual method that uses filter paper disks to diffuse antimicrobials Gradient diffusion: A manual method 69 Antimicrobial Susceptibility Tests Fig. 15.26. Agar disk diffusion. (A) Mueller-Hinton agar has been inoculated with the test organism, and the antibiotic disks have been added to the media. No incubation has yet occurred. (B) After 16 to 18 hours incubation, the zones of inhibition are apparent. The zones of inhibition are measured and compared to a table of values for each antibiotic. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 70 Antimicrobial Susceptibility Tests Antimicrobial susceptibility test results are usually reported as susceptible, intermediate, or resistant: Susceptible The antibiotic was effective in killing or stopping the growth of the bacteria or fungus. This is the preferred treatment option. Intermediate The antibiotic may be effective at a higher dose or more frequently. It may also only be effective in certain parts of the body. Resistant The antibiotic was not effective in killing or stopping the growth of the bacteria or fungus. This is not a good treatment option. The results are based on the minimum inhibitory concentration (MIC), which is the lowest concentration of an antibiotic that will stop the growth of bacteria. The laboratory will usually include an interpretation of the results in addition to the MIC. 71 72  Quality Control in the Microbiology Laboratory Quality control (QC) in a microbiology laboratory ensures the accuracy of test results and the reliability of equipment, media, reagents, and specimens: Equipment: Equipment should be consistent each time it's used. Laboratories should calibrate and maintain equipment and ensure it's clean and sterile. Media: Culture media should be tested for sterility and performance. Reagents: Chemicals should be sterilized to prevent contamination. Specimens: Specimens should be collected, transported, and processed properly. Antimicrobial tests: Laboratories should perform antimicrobial susceptibility testing accurately. This includes reviewing the profile for each organism tested before reporting results. 73  Tests for Fungi (Mycology) Fungi are yeasts and molds. Characteristics of fungi ◦ Fungi are eukaryotes, possessing a true nucleus with a nuclear membrane and mitochondria, whereas bacteria are prokaryotes, lacking these structures. ◦ Fungal cell walls are not composed of peptidoglycan found in bacterial cell walls, but contain alternate carbohydrates such as chitin. ◦ Yeasts are single-celled organisms that reproduce by budding. ◦ Molds have a basic structure that consists of tube-like filamentous projections called hyphae. Hyphae continue to grow, forming an intertwined mass collectively known as mycelia (singular mycelium). 74  Tests for Fungi (Mycology) Fig. 15.27. Pseudohyphae consisting of elongated cells with constrictions at attachment sites. (From Mahon CR, Lehman DC, Manuselis: Textbook of Diagnostic Microbiology, ed. 5, 2015.) 75  Tests for Fungi (Mycology) Tests for fungi can include: Microscopic examination A sample is examined under a microscope, sometimes with a stain or preparation. This can be enough to determine if a fungus is causing an infection. Fungal culture A sample is sent to a lab to grow and identify the fungus. This is the primary test for diagnosing fungal infections. However, it can take several weeks for some fungi to grow. Susceptibility testing A follow-up test to a fungal culture that helps determine how sensitive the fungus is to specific antifungal medications. Antigen and antibody tests Available for some fungi, but only for deep or systemic infections. These tests can be performed on blood or other bodily fluids. MALDI-TOF A rapid sample analysis technique that can identify fungi in minutes. The type of sample collected depends on where the infection is suspected to be. For example, a fungal skin infection might be diagnosed with a skin scraping, hair sample, or nail clipping. A fungal infection in another part of the body might be diagnosed with a sample of blood, urine, sputum, vaginal secretions, or cerebrospinal fluid. 76 Tests for fungi The India ink preparation and the germ tube test are both laboratory methods used to identify fungi: India ink preparation A negative staining technique that detects the capsule of encapsulated fungi, such as Cryptococcus neoformans. The capsule repels the India ink, which appears as a halo around the yeast cell. This test is quick and inexpensive, but it's not very sensitive and can be negative in up to half of cases of cryptococcal meningoencephalitis. Germ tube test A rapid screening test that identifies Candida albicans by its production of germ tubes. The germ tube is a straight-sided extension of the yeast cell that's about half the width and three to four times the length of the cell. The test involves inoculating fetal bovine serum with the yeast isolate and incubating it at 35–37° C for 2.5–3 hours. Some species, like Candida tropicalis, may form pseudohyphae instead of germ tubes, which can lead to false positives. 77  Tests for Fungi (Mycology) Fig. 15.28. Gram stain of yeast. Note: Yeast are not classified as gram positive because of absorption of Gentian Violet stain. (From de la Maza LM, Pezzlo MT, Baron EJ: Color atlas of diagnostic microbiology, St Louis, 1997, Mosby.) 78  Tests for Parasites (Parasitology) Parasites live in or on their hosts, at the expense of the host. ◦ Some parasites cannot survive without their designated host. ◦ Other parasites can exist in a free-living state as an intermediate state before transmission to a suitable host. ◦ Still others live commensally, a situation in which the parasite and the host exist together with no harm coming to the host; the relationship is beneficial to both. Parasites as source of infection ◦ As a result of increased travel to tropical areas, and because of the great influx of refugee populations, many organisms endemic elsewhere are being identified in patients in the United States. 79  Tests for Parasites (Parasitology) Collection of specimens for parasite identification ◦ Stool specimens-A stool exam, also known as an ova and parasite (O&P) test, is a common way to identify parasites in the digestive tract ◦ Blood specimens-making thin/thick smears-Thick blood smears are more effective at detecting parasites because they examine a larger sample of blood. Thin blood smears help identify the species of parasite causing the infection. For example: babesia Methods for detection of parasites ◦ Wet mount, direct smear-Parasites have distinct shapes, sizes, and internal structures (PINWORM test) ◦ PCR can detect the DNA or RNA of parasites in stool samples 80  Tests for Parasites (Parasitology) Common parasites identified ◦ Trichomonas vaginalis ◦ T. vaginalis can inhabit the urogenital system of both males and females. ◦ It is considered a pathogenic parasite; T. vaginalis is the cause of vaginitis, urethritis, and prostatitis. Rapid antigen test: A quick test that can be performed at a provider's office and usually produces results in under 15 minutes Culture test: A sample is sent to a lab where cells grow for up to a week, and then examined under a microscope Nucleic acid amplification testing (NAAT): A laboratory method that identifies the parasite's genetic material 81  Tests for Parasites (Parasitology) Fig. 15.34. Trichomonas vaginalis trophozoite. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 82  Tests for Parasites (Parasitology) Fig. 15.36. Cellophane tape preparation of Enterobius vermicularis (pinworm) eggs. (From Tille PM: Bailey and Scott’s diagnostic microbiology, ed 13, St Louis, 2014, Mosby.) 83 Additional Point-of-Care Rapid Testing Applications Rapid testing for streptococcal infections Rapid testing for pneumococcus and legionellae Rapid for plasmodium falciparum—falciparum malaria (parasites) 84 Automation MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry is a technique that measures the mass of molecules in a sample. It's used in many fields, including: Microbiology: MALDI-TOF MS is a common tool for identifying bacteria, fungi, and other microbes in clinical laboratories. It's faster, more accurate, and less expensive than traditional biochemical identification methods. 85