Microbiology: Basic and Clinical Principles Chapter 7 PDF

Microbiology: Basic and Clinical Principles Second Edition Chapter 7 Fundamentals of Microbial Growth and Decontamination...

Microbiology: Basic and Clinical Principles Second Edition Chapter 7 Fundamentals of Microbial Growth and Decontamination Presented by Janet Dowding, Ph.D. St. Petersburg College Copyright © 2023 Pearson Education, Inc. All Rights Reserved Clinical Case Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbial Growth Basics After reading this section, you should be able to: Discuss basic differences in features of microbial growth in a laboratory versus in nature. Define binary fission and compare it to budding and spore formation. Calculate generation time for a bacterium. Outline the features of the four stages of bacterial growth in a closed batch system. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes Show Dynamic and Complex Growth in Nature, and Often Form Biofilms (1 of 4) When nutritional requirements are met, a microbe will enlarge in size and eventually divide Microbial growth is cell division that produces new (daughter) cells and increases the total cell population Most of our knowledge comes from studying species that can be cultured in the laboratory – of the bacteria species on our planet Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes Show Dynamic and Complex Growth in Nature, and Often Form Biofilms (2 of 4) In the laboratory, bacteria are usually grown as pure, single-species cultures But in nature, bacteria intermingle and live side by side with archaea and eukaryotes Environmental factors play a significant role in the life, metabolism, and structure of bacteria – Escherichia coli converts from a motile bacillus shape to a filamentous nonmotile form during urinary tract infections Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes Show Dynamic and Complex Growth in Nature, and Often Form Biofilms (3 of 4) Cells living in biofilm communities communicate and collaborate to survive In healthcare settings, biofilms are a major concern – Difficult to treat – Contribute to persistent infections Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes Show Dynamic and Complex Growth in Nature, and Often Form Biofilms (4 of 4) Biofilm formation occurs when free-floating (planktonic) bacteria adhere to a surface – Indwelling devices (e.g., catheters, heart valves) are possible havens for biofilms Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria Usually Divide by Binary Fission, but Some May Use Budding or Spore Formation (1 of 4) Binary fission – Occurs in most prokaryotes – Involves dividing a single cell into two cells – Asexual process Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria Usually Divide by Binary Fission, but Some May Use Budding or Spore Formation (2 of 4) The process of binary fission… – Before dividing, the chromosome is replicated – Parent cell begins to pinch off at the middle – Partition (septum) in the center becomes complete – Creates two genetically identical daughter cells Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria Usually Divide by Binary Fission, but Some May Use Budding or Spore Formation (3 of 4) Budding – Asexual reproduction – Original cell elongates then develops a small outgrowth on one side – Chromosome is duplicated and placed in the bud – Separation from the mother cell occurs – Performed by certain fungi and some bacteria (e.g., Hyphomicrobium) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria Usually Divide by Binary Fission, but Some May Use Budding or Spore Formation (4 of 4) Spore formation – Performed by some fungi and bacteria ▪ Can be sexual or asexual in fungi ▪ Asexual in bacteria – Formation varies: ▪ Streptomyces form spores that hang off of long hyphae extensions ▪ Bacterial endospores are thick-walled, nongrowing structure Copyright © 2023 Pearson Education, Inc. All Rights Reserved Generation Time (1 of 3) Generation time – Time it takes for a cell to divide – Times are diverse ▪ Range from about 15 minutes to 24 hours ▪ Depends on the species and conditions Copyright © 2023 Pearson Education, Inc. All Rights Reserved Generation Time (2 of 3) As bacteria divide by binary fission they exhibit exponential growth QUESTION: E. coli are grown for one hour. During that time, they go through three generations of growth. What is the generation time? Growth time (in minutes) Generation time (in minutes) = Number of generations 60 E. coli cell Generation time = minutes 3 in culture ANSWER: Generation time = 20 minutes 1st 2nd 3rd generation 1 hour (or 60generation minutes) generation Copyright © 2023 Pearson Education, Inc. All Rights Reserved Generation Time (3 of 3) The nutrients available impact how fast a microbial population increases – Generation time for many common bacteria is less than an hour ▪ E. coli: 20 minutes – Some bacteria have fairly slow generation times ▪ Mycobacterium tuberculosis: 15–20 hours Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria have Four Distinct Growth Phases When Cultured Using a Closed Pure Batch System (1 of 4) In the lab, bacteria are usually isolated and Cells adjust Exponential Number of cells Cells die as waste accumulates to their Cell dividing = and nutrients grown in closed pure environment growth number of cells dying are depleted batch cultures Buildup of waste and dead cells Bacteria undergoes Stationary phase distinct growth phases Log phase Death phase that can be detected by NUMBER OF VIABLE CELLS counting the number of (LOG) viable cells Lag phase TIME (HOURS) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria have Four Distinct Growth Phases When Cultured Using a Closed Pure Batch System (2 of 4) Phase One: Lag Phase – Delay that occurs while cells adjust to their new environment Phase Two: Log Phase – Period of rapid exponential growth Phase Three: Stationary Phase – Nutrients are depleted, waste accumulates – Population growth rate levels off Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria have Four Distinct Growth Phases When Cultured Using a Closed Pure Batch System (3 of 4) Phase Four: Death Phase – At a critical point of waste buildup and decreasing nutrients, the cells begin to die – Rate of cell death is exponential – Small number of the cells survive by adapting to the waste and by feeding off dead cells Copyright © 2023 Pearson Education, Inc. All Rights Reserved Bacteria have Four Distinct Growth Phases When Cultured Using a Closed Pure Batch System (4 of 4) In industry, maintaining cells at a specific growth phase is often necessary Chemostat – Fresh growth medium is added – Waste and excess cells are removed Air in Air out Feeder tube delivers fresh media Exit tube removes waste Cells and cells growing in media Stirrer Chemosta t Copyright © 2023 Pearson Education, Inc. All Rights Reserved Prokaryotic Growth Requirements (1 of 2) After reading this section, you should be able to: Define the terms optimal, minimum, and maximum as they apply to temperature and pH conditions. Describe the temperatures where psychrophiles, psychrotrophs, mesophiles, thermophiles, and extreme thermophiles would thrive, and state the grouping for most pathogens. Define acidophiles, alkaliphiles, and neutralophiles and describe ways that microbes survive in pH extremes. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Prokaryotic Growth Requirements (2 of 2) After reading this section, you should be able to: Define the term halophile and state how these microbes combat osmotic stress. Name the various classes of microbes according to their oxygen use and tolerance. Define the terms essential nutrient and growth factor, then describe the energy sources required by phototrophs versus chemotrophs. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Prokaryotes Adapt to Various Growth Conditions All microbes find a niche by adapting to specific conditions (e.g., temperature, pH, salinity, levels of oxygen, and available nutrients) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (1 of 6) Temperature is an important component of a microbe’s environment – Low temperature ▪ Decrease enzymatic reactions – Increased temperature ▪ Speeds up enzymatic reactions ▪ Can increase growth rate – High temperatures ▪ Denature cell proteins (kills cell) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (2 of 6) Three principal temperatures: – Maximum temperature —highest temperature that supports growth – Minimum temperature —lowest temperature that supports growth – Optimal temperature — temperature where cellular growth is highest Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (3 of 6) Temperature is a ºCelsius fundamentally important 130 120 factor that can be used to 110 100 100ºC (water boils) classify microbes Extreme thermophiles: 90 65–120ºC 80 70 Thermophiles: 60 40–75ºC 50 40 Mesophiles: 37ºC 10–50ºC 30 (human body temperature) 20 20ºC Psychrotrophs: (room temperature) 0–30ºC 10 0 0ºC Psychrophiles: (water freezes) –20–10ºC –10 –20 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (4 of 6) Psychrophiles – Thrive between Psychrotrophs – Grow at about – Associated with foodborne illness Mesophiles – Grow best around – Associated with most pathogens Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (5 of 6) Thermophiles – Grow around – Associated with compost piles and hot springs Extreme thermophiles – Grow around Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature (6 of 6) High-temperature environments frequently have extremes in pressure (e.g., thermal vents) Barophiles can withstand the high-pressure environment of the deep sea Copyright © 2023 Pearson Education, Inc. All Rights Reserved pH (1 of 2) Every microbe has a minimum, optimum, and maximum range of pH for growth Acidophiles – Grow at pH 1 (or less) to pH 5 – Live in areas such as sulfur hot springs and volcanic vents – Often maintain a fairly neutral cytoplasmic pH – Proton pumps export excess protons from the cytoplasm to raise pH Copyright © 2023 Pearson Education, Inc. All Rights Reserved pH (2 of 2) Neutralophiles – Grow best in a pH range of 5–8 – Make up the majority of microorganisms Alkaliphiles – Grow in the basic pH range of 9–11 – Associated with soda lakes Copyright © 2023 Pearson Education, Inc. All Rights Reserved High-Salt Conditions (1 of 3) Halophiles – Thrive in high-salt environments – Tolerate up to 35% – Associated with the Dead Sea and the Great Salt Lake of Utah Facultative halophiles – Tolerate higher salt but may not grow well – Example: Staphylococcus aureus Copyright © 2023 Pearson Education, Inc. All Rights Reserved High-Salt Conditions (2 of 3) Bacterial cytoplasm is 80% water Normal cells undergo plasmolysis (due to osmosis) Space between cell wall and plasma membrane Copyright © 2023 Pearson Education, Inc. All Rights Reserved High-Salt Conditions (3 of 3) Halophiles must overcome the osmotic stress of a high-salt environment – Keep high concentrations of organic materials and ions in their cytoplasm Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (1 of 7) Many microbes on this planet live either without oxygen or with minimal oxygen Oxygen levels are low beneath the soil or within silt deposits in lakes and oceans Most pathogens thrive in low-oxygen environments within the host Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (2 of 7) Atmospheric oxygen easily diffuses across cell plasma membranes Inside the cell, some of the oxygen is converted into reactive oxygen species (ROS) – Superoxide ions – Hydrogen peroxide ROS can rapidly damage proteins and DNA Many microbes have evolved ways to detoxify ROS (e.g., aerobes) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (3 of 7) Many aerobic microbes rely on antioxidants (compounds and enzymes) to detoxify ROS – Superoxide dismutase converts reactive superoxide ions to hydrogen peroxide – Catalase converts the hydrogen peroxide to water and oxygen Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (4 of 7) Obligate aerobes – Absolute dependence on for cellular processes Microaerophiles – Use only small amounts of – Live in low settings Facultative anaerobes – Grow with and without – Switch between using and fermentation Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (5 of 7) Anaerobes – Do not use in their metabolic processes Aerotolerant anaerobes – Tolerate but don’t use it in their metabolic processes – Have ways to deactivate ROS Obligate anaerobes – Do not use in their metabolism – Can’t eliminate ROS – Tend to die in aerobic environments Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (6 of 7) Table 7.1 Oxygen Use and Tolerance Classifications Blank Obligate Obligate Microaerophi Aerotolerant Facultative Aerobe Anaerobe le Anaerobe Anaerobe Appearance of growth reflects oxygen tolerance/use. Medium is semisolid thioglycolate, which generates an oxygen gradient The figure illustrates a plugged test tube filled up to three-fourths with a medium that consists of spherical cells floating on the surface of the medium. The figure illustrates a plugged test tube filled up to three-fourths with a medium that consists of spherical cells clustered at the bottom of the test tube. The figure illustrates a plugged test tube filled up to three-fourths with a medium that consists of spherical cells floating in the middle region of the test tube. The figure illustrates a plugged test tube filled up to three-fourths with a medium that consists of spherical cells floating throughout the medium. The figure illustrates a plugged test tube filled up to three-fourths with a medium that consists of spherical cells floating throughout the medium with dense clusters on the surface. ranging from an oxygen-rich environment at the very top to an anaerobic environment at the bottom. Oxygen use in metabolism Absolute Not used Small Not used Prefer using dependence amounts oxygen but can survive without it Can effectively manage reactive Yes No Yes (but only Yes Yes oxygen species? low amounts) *Semisolid thioglycolate media is used to generate an oxygen gradient ranging from an oxygen-rich environment at the very top of the media to an anaerobic environment at the bottom. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Oxygen Requirements (7 of 7) Examples of pathogens and their tolerance Lungs Skin Examples: Bordetella pertussis Examples: (whooping cough): Staphylococcus aureus Obligate aerobe (staph infections): Mycobacterium tuberculosis: Facultative anaerobe Facultative anaerobe Propionibacterium acnes: Mycoplasma pneumoniae: Aerotolerant anaerobe Obligate aerobe Stomach Blood and Lymph Example: Examples: Helicobacter pylori Borrelia burgdorferi (ulcers): (Lyme disease): Microaerophil Microaerophil e e Treponema pallidum (syphilis): Microaerophil e Yersinia pestis Large Intestine (plague): Facultative anaerobe Examples: Clostridioides difficile: Obligate anaerobe Salmonella species: Facultative anaerobe Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes Require Nutrients, Growth Factors, and a Source of Energy Microbes use nutrients from the environment to divide, and to build structural components, enzymes, and other factors Copyright © 2023 Pearson Education, Inc. All Rights Reserved Essential Nutrients (1 of 4) About 90% of a cell’s dry weight is: – Carbon, hydrogen, nongaseous oxygen, and nitrogen Other important elements include: – Sulfur, phosphorus, potassium, sodium, calcium, magnesium, chlorine – Various metal ions (e.g., copper, zinc, iron) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Essential Nutrients (2 of 4) Essential nutrients – Required to build new cells – Found in the organic and inorganic compounds of a microbe’s environment – Macronutrients ▪ Needed in large amounts (e.g., carbon) – Micronutrients ▪ Needed in very small amounts (e.g., iron) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Essential Nutrients (3 of 4) There are two categories of organisms based on how they obtain organic carbon: – Heterotrophs require an external source of organic carbon (e.g., sugars, lipids, proteins) – Autotrophs do not require an external source of organic carbon ▪ Use carbon fixation to convert inorganic carbon into organic carbon Copyright © 2023 Pearson Education, Inc. All Rights Reserved Essential Nutrients (4 of 4) Most of a cell’s nitrogen and phosphorus are extracted from organic nutrients Some cells get their nitrogen directly from the atmosphere (nitrogen fixation) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Growth Factors (1 of 2) Some microbes can’t build all of their organic precursors (e.g., amino acids, vitamins, nitrogenous bases) Cells must import these required substances from their environment The necessary substances that a cell can’t make on its own are called growth factors The organism will not grow if they are missing from the environment Copyright © 2023 Pearson Education, Inc. All Rights Reserved Growth Factors (2 of 2) Organisms that need multiple growth factors are said to be fastidious When growing fastidious microbes in the laboratory, amino acids, vitamins, and/or nitrogenous bases must be supplied in the growth medium Copyright © 2023 Pearson Education, Inc. All Rights Reserved Energy Sources (1 of 2) In order to carry out functions and construction, cells require energy: – Phototrophs are organisms that use light energy – Chemotrophs are organisms that break down chemical compounds for energy Copyright © 2023 Pearson Education, Inc. All Rights Reserved Energy Sources (2 of 2) PHOTOTROPHS CHEMOTROPHS Energ Sunlight Nutrient y breakdow sourc n e Photoautotrop Photoheterotrop Chemoautotrop Chemoheterotroph h h h Carbo Inorganic Organic Inorganic Organic n (usually CO2) (usually CO2) source Exampl Cyanobacteria Heliobacillus Thiobacillus Escherichia coli, a e found in mobilis denitrificans common inhabitant freshwater found in rice found in soil, mud, of environments paddy and mammalian fields freshwater and intestines marine sediments Copyright © 2023 Pearson Education, Inc. All Rights Reserved Growing, Isolating, and Counting Microbes After reading this section, you should be able to: Describe the three formats of media and state when each would be used. Explain the features and uses of complex, defined, selective, and differential media. Provide examples of how to culture anaerobic microbes. Describe the considerations made in collecting clinical samples. Summarize the streak plate technique and state its purpose. Describe direct and indirect methods for cell enumeration. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Microbes are Grown Using Various Media An assortment of culture media help us grow microbes We classify media by their physical state (liquid, solid, or semisolid formats), their chemical composition, and their function Copyright © 2023 Pearson Education, Inc. All Rights Reserved Physical State: Liquid, Solid, and Semisolid Media (1 of 4) The physical state, or format, of media used depends on the application – Liquid media (i.e., broth media) are ideal for growing large batches of microbes – Solid media are useful for isolating colonies and observing specific culture characteristics – Semisolid media is useful for motility testing Copyright © 2023 Pearson Education, Inc. All Rights Reserved Physical State: Liquid, Solid, and Semisolid Media (2 of 4) Broth media – Made by adding various nutrients to purified water – Poured into flasks or tubes and sterilized Copyright © 2023 Pearson Education, Inc. All Rights Reserved Physical State: Liquid, Solid, and Semisolid Media (3 of 4) Solid and semisolid media in Petri plates – Made by adding a powdered polysaccharide called agar to liquid media ▪ Semisolid media contain less agar than solid media – Medium is heat sterilized – While hot, the medium is poured into petri plates – Allowed to cool and solidify Copyright © 2023 Pearson Education, Inc. All Rights Reserved Physical State: Liquid, Solid, and Semisolid Media (4 of 4) Solid and semisolid media in slants and tubes – Medium is heat sterilized – While hot, the medium is poured into tubes – Allowed to cool and solidify ▪ Slants cool at an angle ▪ Deeps cool upright Simmons citrate Motility test test Copyright © 2023 Pearson Education, Inc. All Rights Reserved Chemical Composition (Complex and Defined Media) (1 of 3) Based on their chemical composition, media can be described as defined or complex Table 7.2 Complex and Defined Media Examples Complex Media Defined Media Example: Luria–Bertani Media (Ingredients Example: Glucose Minimal Salts Media (Ingredients in 1 Liter of in 1 Liter of Liquid Medium) Liquid Medium) 10 grams of tryptone (broken-down milk 20 mL of a 20% glucose solution protein) 2 mL of 1 molar magnesium sulfate M g S O sub 4 5 grams of yeast extract (ground-up yeast 0.1 mL of 1 molar calcium chloride C a C l sub 2 cells) 12.8 g of disodium phosphate N a sub 2 H P O sub 4 10 grams of sodium chloride (N aCl) 3.0 g of potassium phosphate K H sub 2 P O sub 4 Water added to bring level to 1L 0.5 g of sodium chloride (N aCl) 1.0 g of ammonium chloride N H sub 4 C l Water added to bring level to 1L Although plenty of amino acids and sugars Growth factors like vitamins and amino acids are not added to minimal are provided in the complex yeast and protein slats media, so any organism grown on it must be able to take the preparations, we don’t know the exact levels nitrogen supplied in the ammonium chloride and use it to build all of each. necessary amino acids. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Chemical Composition (Complex and Defined Media) (2 of 3) Defined media (also called synthetic media) – Chemically defined or precisely known composition – Each organic and inorganic component is completely known and quantified – Useful for growing certain autotrophs and some heterotrophs Copyright © 2023 Pearson Education, Inc. All Rights Reserved Chemical Composition (Complex and Defined Media) (3 of 3) Complex media (also called enriched media) – Contains a mixture of organic and inorganic nutrients that are not fully defined – Contain more complex ingredients (e.g., blood, milk proteins, extracts) – Precise quantity of every vitamin and nutrient is unknown – Used to grow fastidious organisms with complex growth requirements Copyright © 2023 Pearson Education, Inc. All Rights Reserved Function (Differential and Selective Media) Clinical samples – Urine sample, throat swab, and fecal (stool) – NOT pure cultures – Must be able to separate out pathogens from among the normal microbiota – Helpful tools to use selective and differential media Copyright © 2023 Pearson Education, Inc. All Rights Reserved Differential Media (1 of 2) Differential media – Media formulated to visually distinguish one microbe from another Copyright © 2023 Pearson Education, Inc. All Rights Reserved Differential Media (2 of 2) Common example of a differential medium is blood agar – Beta α β γ hemolytic—break down red blood cells – Alpha hemolytic—partial break down of red blood cells Blood agar inoculated with bacteria that exhibit – Gamma alpha, beta, and gamma hemolysis. hemolytic—do not lyse red blood cells Copyright © 2023 Pearson Education, Inc. All Rights Reserved Selective Media (1 of 3) Selective media – Single out bacteria that have specific properties – Ingredients foster the growth of certain bacteria and suppress the growth of others – Examples: ▪ Mannitol salt agar (MSA) ▪ Eosin methylene blue agar (EMB) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Selective Media (2 of 3) Mannitol salt agar (MSA) – Selective due to its high salt content – Differentiates organisms based on their ability to ferment a sugar called mannitol Mannitol Mannitol fermente not d fermented Mannitol salt agar Copyright © 2023 Pearson Education, Inc. All Rights Reserved Selective Media (3 of 3) Eosin methylene blue agar (EMB) – Dyes eosin and methylene blue limit Lactose Lactose Gram-positive fermented (strong not fermented bacterial growth acid made) – Differentiates based Lactose on ability to ferment fermente d lactose Eosin methylene blue agar Copyright © 2023 Pearson Education, Inc. All Rights Reserved Anaerobic Media (1 of 4) On average, anaerobes make up of the bacterial population in a wound Clinicians must be careful when collecting anaerobic samples – Exposure to can kill the microbes before they can be cultured To prevent this, samples are quickly deposited and capped in specialized transport media tubes Copyright © 2023 Pearson Education, Inc. All Rights Reserved Anaerobic Media (2 of 4) Molecular oxygen is removed from media in a number of ways – Thioglycate is added to media ▪ Reducing agent; converts O2 to water – Anaerobic jar is used ▪ Sample is added to chamber ▪ Packet of oxygen-reacting chemicals is opened inside it, creating oxygen-free conditions Copyright © 2023 Pearson Education, Inc. All Rights Reserved Anaerobic Media (3 of 4) – Anaerobic chamber is used ▪ Large anaerobic box ▪ Gloves are inserted into the chamber to allow handling of organisms ▪ Samples are placed in a side compartment ▪ Nitrogen are piped into the chamber to displace all oxygen Copyright © 2023 Pearson Education, Inc. All Rights Reserved Anaerobic Media (4 of 4) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Collecting, Isolating, Counting, and Identifying Microbes are Important in Microbiology Aseptic techniques – Methods designed to prevent introducing contaminating microbes to a patient, a clinical sample, or others in the healthcare setting – Protect people and promote sample integrity Healthcare workers apply these techniques as a routine part of patient care when administering an injection or collecting a clinical sample for microbiological analysis Copyright © 2023 Pearson Education, Inc. All Rights Reserved Collecting Samples for Clinical Microbial Analysis (1 of 2) All clinical samples must be collected and transported aseptically to prevent contamination Requires samples to be collected using sterile materials Collection containers used depend on the tests that need to be done Proper hand washing is essential before and after specimen collection Copyright © 2023 Pearson Education, Inc. All Rights Reserved Collecting Samples for Clinical Microbial Analysis (2 of 2) Wear gloves during sample collection Quickly seal samples in containers Properties of the suspected causative agent must also be considered during collection Collected sample may have to be immediately refrigerated or placed on dry ice to ship Copyright © 2023 Pearson Education, Inc. All Rights Reserved Isolating Microbes Using the Streak Plate Technique (1 of 3) To identify the potential pathogen in a clinical sample it must first be isolated Streak plate technique is the most commonly used technique to isolate bacteria – Method dilutes a culture on an agar plate – Individual cells are thinly separated from one another over the medium’s surface – As cells divide, their population increases to form a mound of cells called a colony Copyright © 2023 Pearson Education, Inc. All Rights Reserved Isolating Microbes Using the Streak Plate Technique (2 of 3) 3 2 1 Copyright © 2023 Pearson Education, Inc. All Rights Reserved Isolating Microbes Using the Streak Plate Technique (3 of 3) If multiple species are growing on the plate it is possible to see a mixture of colonies that exhibit a range of shapes, colors, sheen, and textures Copyright © 2023 Pearson Education, Inc. All Rights Reserved Methods for Counting Microbes Sometimes it’s important to determine how many microbes there are in a sample Food and beverage manufacturers and water treatment plants must conduct microbial counts to ensure product quality Clinical laboratories may use bacterial growth rates from patient samples to determine antibiotic susceptibility Microbes can be enumerated using direct or indirect methods Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (1 of 7) These methods involve counting individual cells or colonies (plate counts) Cell count enumerates the number of cells in a small portion of the sample Cell counts can be done using automated or manual procedures Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (2 of 7) Manual cell counting Cover slip requires: – Microscope – Specialized Culture added here Counting counting chamber chamber that has a slide volumetric grid etched on it Cells in the grid are counted using a microscope Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (3 of 7) Highly reproducible, automatic counting methods can be used to count the cells in a culture Coulter counter is a machine that counts the number of cells as they pass through a thin tube Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (4 of 7) Suspension of stained cells Flow cytometer uses a laser light to detect cells passing through a narrow channel Nozzle Fluorescent labeled cells – Cells are fluorescently labeled before counting Fluorescence from stained cells – Ability to differentiate Scattered Light source one cell type from light from all cells Detecto r another by using different colored labels Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (5 of 7) Viable plate count allows for direct enumeration of bacteria using agar plates – Samples are serially diluted – Applied to agar using either spread plate method or pour plate method – After an incubation period, colonies are visible and can be counted Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (6 of 7) Original sample is sequentially diluted Nondilut Dilute e 1.0 1.0 1.0 mL mL mL Initial 9 mL 9 mL 9 mL sample brot brot brot h h h 0.1 mL of diluted samples is then spread on plates (spread plate method) or 1.0 mL is poured into plates and mixed with melted agar (pour plate method) Colonies Following incubation, colonies are counted Copyright © 2023 Pearson Education, Inc. All Rights Reserved Direct Methods (7 of 7) – Taking the dilution factor into consideration, the total number of living cells are calculated – Numerical data for plate counts is usually represented as: ▪ Colony-forming units (CFU) per milliliter (or per gram) ▪ Reflects that sometimes a clump of cells give rise to a colony Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect Methods (1 of 3) Indirect methods rely on secondary reflections of overall population size Fast and easy way to indirectly measure cell numbers is to measure turbidity – More cells = cloudier (more turbid) – Spectrophotometer measures either transmission or absorbance (optical density) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect Methods (2 of 3) Light-sensitiv Direct e light detector Light source Percent light transmitted Spectrophotomete Uninoculated r tube Scattered light that does not reach detector Light source Percent light transmitted Spectrophotomete Inoculated r broth culture Copyright © 2023 Pearson Education, Inc. All Rights Reserved Indirect Methods (3 of 3) Other indirect methods for enumeration include: – Assessing total dry weight – Detecting the levels of metabolic activity in a sample Copyright © 2023 Pearson Education, Inc. All Rights Reserved Table 7.3 Table 7.3 Direct and Indirect Cell-Counting Methods Method Name Description Direct Microscopic count Manual count of cells using a microscope and counting chamber; doesn’t differentiate between living or dead cells Direct Coulter counter or flow Both are automated methods; Coulter counter doesn’t cytometer differentiate between living or dead cells. Flow cytometer distinguishes cells by detecting fluorescent tags, and can differentiate between living and dead cells Direct Viable plate count Colonies that grow from a diluted cell sample account for original cell count Indirect Turbidity measurement Spectrophotometer is used to measure cloudiness of a liquid culture Indirect Dry weight Dry mass measurement Indirect Biochemical activity Byproducts of cell growth are measured Copyright © 2023 Pearson Education, Inc. All Rights Reserved Methods for Identifying Microbes Physical, biochemical, and genetic analysis are all commonly used tools to identify microbes – Physical analysis involves staining and microscopy to observe morphological features – Biochemical analysis involves a collection of media that assess metabolic properties – Genetic methods also help to quickly identify microbes ▪ Probes, polymerase chain reaction (PCR), DNA “fingerprinting,” electrophoresis separation methods Copyright © 2023 Pearson Education, Inc. All Rights Reserved Controlling Microbial Growth (1 of 2) After reading this section, you should be able to: Define the terms decontamination, sterilization, disinfection, microbiostatic, microbiocidal, disinfectant, and antiseptic. Describe different heat treatments used to control microbe levels. State when each is used, and define decimal reduction time, thermal death point, and thermal death time. Describe radiation and filtration controls and state when each is used. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Controlling Microbial Growth (2 of 2) After reading this section, you should be able to: Name and describe the various classes of germicides and discuss what goes into selecting the right one for a given situation. Name the levels of germicide activity and identify the types used for critical, semicritical, or noncritical equipment. Discuss the factors that should be considered when selecting an appropriate germicide. Provide examples of how Mycobacteria, endospore, virus, protozoan, and prion levels are each controlled. Copyright © 2023 Pearson Education, Inc. All Rights Reserved Control Strategies Aim to Reduce or Eliminate Microbial Contamination (1 of 2) Microbial control measures occur in every area of our lives (e.g., water sanitation to hospital and restaurant cleaning standards) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Control Strategies Aim to Reduce or Eliminate Microbial Contamination (2 of 2) Decontamination removes or reduces microbial populations to render an object safe for handling Sterilization eliminates all bacteria, viruses, and endospores – Required for drugs, objects used for medical procedures, and for lab media and glassware Disinfection reduces microbial numbers – Use for cosmetics, foods, surfaces, and external medical equipment Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature, Radiation, and Filtration are All Physical Methods to Control Microbial Growth Physical controls most commonly include: – Temperature changes (heat/cold) – Radiation – Filtration Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature Changes (1 of 3) Both cold and heat have important roles in controlling microbial growth Refrigeration and freezing slow the growth of microbes – Slows food spoilage – In the lab, used to preserve specimen isolates and increase the shelf life of media – Refrigeration preserves clinical samples Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature Changes (2 of 3) Most microbes are sensitive to heat Heat can be used to achieve either sterilization or decontamination Decimal reduction time (DRT or D value) – Time in minutes it takes to kill 90% of a given microbial population at a set temperature – Associated with disinfection Copyright © 2023 Pearson Education, Inc. All Rights Reserved Temperature Changes (3 of 3) Sterilization has temperature-related parameters... Thermal death time – Shortest period of time at a certain temperature needed to kill all microbes in a sample Thermal death point – Minimum temperature needed to kill all microbes in a sample within 10 minutes Copyright © 2023 Pearson Education, Inc. All Rights Reserved Autoclaving (1 of 2) Steam Autoclave is a machine exhaust pipe that applies steam heat Inner chamber along with pressure to Steam in Door sterilize chamber from jacket – Used for Jacke t microbiological media To drain Steam supply and assorted medical or lab equipment Copyright © 2023 Pearson Education, Inc. All Rights Reserved Autoclaving (2 of 2) Most substances are sterile within 20 minutes using standard autoclave settings – Pressure of 15 pounds per square inch – Steam heat at 121 oC Copyright © 2023 Pearson Education, Inc. All Rights Reserved Boiling Another way to reduce microbial numbers is through boiling Municipalities often issue a “boil water advisory” when drinking water is contaminated Boiling water for 5 minutes eliminates most pathogenic bacteria, protozoans, and viruses Endospores can withstand hours of boiling therefore it is not an efficient sterilization method Copyright © 2023 Pearson Education, Inc. All Rights Reserved Pasteurization Pasteurization is used to eliminate pathogens (e.g. Listeria, Salmonella, E. coli O157:H7) – Application of moderate heat (below the liquid’s boiling point) – Eliminates pathogens and reduces harmless microbes that cause milk spoilage Copyright © 2023 Pearson Education, Inc. All Rights Reserved Dry Heat Incineration or hot-air ovens can also be used for sterilization or disinfection Common examples of dry heat sterilization: – Heating an inoculating loop to red hot in a Bunsen burner flame – Incinerating waste – Placing an object at for 2 hours in a dry heat oven achieves sterilization Copyright © 2023 Pearson Education, Inc. All Rights Reserved Radiation Some physical decontamination methods involve radiation, or high-energy waves Radiation can serve as a disinfection or sterilization tool depending on the protocol Radiation is either ionizing or nonionizing Copyright © 2023 Pearson Education, Inc. All Rights Reserved Ionizing Radiation Ionizing radiation – Gamma rays and X-rays – Generate reactive ions that kill microbes and inactivate viruses – Damage nucleic acids Ionizing radiation passes though packaging – Useful in food and pharmaceutical industries – Sterilizes medical supplies that can’t be autoclaved Copyright © 2023 Pearson Education, Inc. All Rights Reserved Nonionizing Radiation (1 of 2) Nonionizing radiation – Ultraviolet (UV) rays – Causes thymine dimers – Alter structure of DNA leading to mutation Copyright © 2023 Pearson Education, Inc. All Rights Reserved Nonionizing Radiation (2 of 2) Nonionizing radiation uses: – UV light boxes in air-handling systems – Sanitize drinking water and swimming pools – Disinfect surfaces in operating rooms – Disinfect biosafety cabinet surfaces Doesn’t pass through packaging Copyright © 2023 Pearson Education, Inc. All Rights Reserved Filtration (1 of 4) Large volumes of liquid or air can be passed through microbe-capturing filters Filter pore sizes can even be made small enough to remove viruses High-efficiency particulate air (HEPA) filters remove microbes and allergens from the air Copyright © 2023 Pearson Education, Inc. All Rights Reserved Filtration (2 of 4) HEPA filters are made of randomly Filter frame arranged fibers that remove Air flow 99.97% of airborne substances Continuous sheet of – Pores are 0.3 micrometer filter medium or larger – Do not sterilize air Aluminum Filter sheet of separator randomly arranged fibers Copyright © 2023 Pearson Education, Inc. All Rights Reserved Filtration (3 of 4) Liquids can be sterilized using membrane filters – Pore sizes range from to filter out bacteria and protists, to to remove viruses Process of filtration – Large volumes of liquid are pulled through the filter using a vacuum mechanism – Smaller volumes are pushed through syringes with filters attached to the end Copyright © 2023 Pearson Education, Inc. All Rights Reserved Filtration (4 of 4) “LifeStraws” – filters that remove pathogens from drinking water Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (1 of 7) Chemical control of microbial growth involves chemicals called germicides – Germicides that kill microbes are classified as microbiocidal – Germicides that only inhibit microbial growth are microbiostatic Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (2 of 7) Two key classes: – Disinfectants—used to treat inanimate objects – Antiseptics—applied to living tissue Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (3 of 7) Germicides are ranked across three tiers: – Low-level agents destroy bacteria (but not Mycobacterium tuberculosis), fungi, and some viruses, but not endospores – Intermediate-level agents destroy all bacteria (including M. tuberculosis), fungi, and viruses, but not endospores – High-level agents destroy all microbes and endospores Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (4 of 7) Medical equipment that requires regular decontamination is classified in three tiers: – Critical equipment ▪ Comes into contact with sterile body sites or the vascular system; must be sterilized – Semicritical equipment ▪ Comes in contact with mucous membranes or non–intact skin ▪ Should be free of bacteria, fungi, and viruses with low numbers of endospores Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (5 of 7) – Noncritical equipment ▪ Contact patients’ intact skin ▪ Require less stringent disinfection Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (6 of 7) Table 7.4 Germicides for Reducing or Eliminating Microbes Level* Germicide Mode of Action Pros/Cons Low Detergents Target lipid Pros: Cheap, low toxicity, pleasant scent, useable as membranes disinfectant and antiseptic Cons: Activity is decreased in hard water, easily contaminated by Pseudomonas bacteria Intermediate Alcohols: Target proteins Pros: Cheap, easily applied, useable as disinfectant and Isopropanol and lipid antiseptic Ethanol membranes Cons: Flammable, can react with plastics Intermediate Phenols Target proteins Pros: Easy to apply, effective in hard water, useable as and lipid disinfectant and antiseptic membranes Cons: Leave residue, irritants, harsh on surfaces, medicinal scent, sensitive to water hardness High Aldehydes: Target proteins, Pros: Achieve sterility at certain concentrations Formaldehyde nucleic Cons: Toxic, irritants, and leave a residue Glutaraldehyde acids High Halogens: Oxidizing agents, Pros: Sterilants at higher concentrations, cheap, useable as Chlorine mainly target disinfectant and antiseptic Iodine proteins, nucleic Cons: Rapidly inactivated by organic material, corrosive, acids discolor fabrics Copyright © 2023 Pearson Education, Inc. All Rights Reserved Germicides are Chemical Controls that Limit Microbes (7 of 7) Table 7.4 [continued] Level* Germicide Mode of Action Pros/Cons High Peroxygens: Oxidizing agents that Pros: Effective sterilization at high Hydrogen mainly target nucleic concentrations, useable as disinfectant and peroxide acids and proteins antiseptic; peracetic acid is effective despite Peracetic acid organic material present and has no residue Cons: Most readily inactivated by organic matter, corrosive, irritants High Ethylene oxide Target proteins, Pros: Can treat items that can’t withstand heat nucleic acids or moisture, gentle on equipment Cons: Toxic and flammable *Denotes the highest possible germicide level of the agent. Any germicide that is greatly diluted or improperly applied will have a low effect. Higher concentrations usually provide a higher disinfection potential (or even sterilize). Copyright © 2023 Pearson Education, Inc. All Rights Reserved Alcohols Alcohols – Intermediate-level disinfectants – Denature proteins and attack lipid membranes – Example: ethanol and isopropanol ▪ Optimal concentration is 60–90% – Used to disinfect small equipment (e.g., thermometers, scissors, stethoscopes) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Aldehydes Aldehydes – High- or intermediate-level disinfectants based on concentration – Reacting with proteins and nucleic acid – Example: formaldehyde and glutaraldehyde – Used to sterilize surgical instruments, endoscopes, dialyzers, anesthesia and respiratory equipment Copyright © 2023 Pearson Education, Inc. All Rights Reserved Phenols Phenols – Intermediate-level germicides – Destroy bacterial cell walls and interact with proteins – Example: Used in common disinfectants such as Lysol – Used in personal hygiene items (e.g., soap, mouthwash) as well as clinical use Copyright © 2023 Pearson Education, Inc. All Rights Reserved Halogens (Chlorine and Iodine) Halogens – Oxidize cell components – Example: chlorine and iodine compounds ▪ Chlorine bleach (sodium hypochlorite) is one of the most widely used halogen disinfectants – Used on medical equipment and floors and added to drinking water Copyright © 2023 Pearson Education, Inc. All Rights Reserved Peroxygens Peroxygens – High-level germicides at high concentrations – Can be used as antiseptics and disinfectants – Strong oxidizing properties – Examples: hydrogen peroxide, peracetic acid Copyright © 2023 Pearson Education, Inc. All Rights Reserved Ethylene Oxide Ethylene oxide – Sterilant – Damages proteins and nucleic acids – Colorless gas – Used for temperature-sensitive materials and equipment susceptible to moisture – Applied to implant devices contain electronic parts or plastic components Copyright © 2023 Pearson Education, Inc. All Rights Reserved Detergents (1 of 2) Detergents – Cleaning agent – Amphipathic molecules – Remove water-soluble and water-insoluble substances – Some detergents damage the lipid envelope of certain viruses and the lipid membrane of certain bacterial cells Copyright © 2023 Pearson Education, Inc. All Rights Reserved Detergents (2 of 2) Based on chemical structure, there are classes of detergents: – Anionic detergents have a negative charge and include soaps ▪ Sodium laureth (or laurel) sulfate is a common ingredient in hand soaps, cosmetics, shampoos, laundry detergents, and household cleaning agents – Cationic detergents have a positive charge and include quaternary ammonium compounds (QACs) ▪ Have bactericidal activity and are sporostatic Copyright © 2023 Pearson Education, Inc. All Rights Reserved Many Factors must be Considered to Select an Appropriate Germicide Item uses Germicide reactivity Germicide concentration and treatment times Types of infectious agents being controlled Presence of organic and inorganic matter Impact of germicide residues on equipment use Germicide toxicity Copyright © 2023 Pearson Education, Inc. All Rights Reserved Different Control Methods Work for Different Microbes (1 of 5) Mycobacterium Control – Mycobacterium species cause tuberculosis and leprosy – Contain cell walls rich in waxy mycolic acids – Spread by airborne droplets – Control measures target reducing airborne particles from infected individuals Copyright © 2023 Pearson Education, Inc. All Rights Reserved Different Control Methods Work for Different Microbes (2 of 5) Endospore Control – Endospores are dormant structures – Can revert to growing (vegetative) cells once favorable growth conditions are restored – Endospores survive drying, radiation, boiling, chemicals, and heat treatments – Most effective way to ensure elimination of endospores is by autoclaving – Other methods include hydrogen peroxide vapor at high heat or sporicides Copyright © 2023 Pearson Education, Inc. All Rights Reserved Different Control Methods Work for Different Microbes (3 of 5) Viral Control – Viruses can be resistant to some measures – Lipids in the viral envelope are sensitive to heat, drying, and detergents ▪ Glutaraldehyde-based detergents are effective at inactivating these viruses – Naked viruses are usually inactivated by chlorine-based agents Copyright © 2023 Pearson Education, Inc. All Rights Reserved Different Control Methods Work for Different Microbes (4 of 5) Protozoan Control – Different stages of a protozoan’s life cycle can resist certain control methods – Variety of treatments are used (e.g., filtration, carbon dioxide, UV, and ozone treatments) Copyright © 2023 Pearson Education, Inc. All Rights Reserved Different Control Methods Work for Different Microbes (5 of 5) Prion Control – Infectious proteins called prions – Withstand autoclaving and chemical sterilization – Surgical devices are reused after autoclaving or chemical sterilization – Prions are eliminated through a combination of chemical treatments and increased temperature and pressure during autoclaving Copyright © 2023 Pearson Education, Inc. All Rights Reserved Visual Summary: Fundamentals of Microbial Growth and Decontamination Microbial Environmental Bacteria mainly use binary fission to divide. Generation Growth time is the time it takes for a particular species of cell to divide. Growth Conditions Temperature, pH, salinity, and oxygen levels impact microbial growth. Bacteria also require various organic and in organic nutrients and growth factors. Temperature ranges for growth Mesophiles: 10–50º C Psychrotrophs: Extreme In closed culture, bacterial populations have four distinct thermophiles: 0–30º Thermophiles: growth phases, with exponential growth in the log phase. Psychrophil 65–120 –20–10ºC C 40–75º es: ºC C Stationary phase Death phase Log phase NUMBER OF Oxygen use/tolerance VIABLE CELLS Oxygen (LOG) concentration High Lag phase TIME (HOURS) Lo w Growing and Isolating Media can be solid, liquid, or semisolid and have defined or complex formulations. Obligat Obligate Microaerophile Facultative Aerotolerant Microbes Some are selective and/or differential. e aerobes anaerobe s s anaerobes anaerobes Controlling Microbial Sterilization and disinfection are achieved using chemical or Growth physical methods. Physical methods Hea t Autoclaving The streak plate isolation technique Mannitol salt agar is one type of Boilin helps to isolate a pure culture. selective and differential media. g Pasteurization Dry heat Radiation Population Ionizing (gamma rays/X-rays) While direct methods count colonies or cells, indirect methods Non-ionizing (UV light) Enumeration rely on secondary reflections of overall population size. Filtration Direct methods Air filters (HEPA filter) Viable plate count Liquid filters (membrane filters) Manual cell counts Coloni Automated cell es counts (coulter counter/flow Viable plate counts cytometer) Manual cell counts Chemical methods (germicides) Disinfectants and antiseptics Indirect methods Alcohol Turbidity (spectrophotometry) s Aldehydes Dry weight Phenols Biochemical Halogens activity Peroxygens Ethylene oxide Spectrophotometr Detergents y Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (1 of 6) Summary of the case: – 3 patients ranging from ages 16–18 years old with upper ear cartilage infections – Signs and symptoms included redness, swelling, and pus – The examining nurse practitioner (NP) determine all 3 patients had recent piercings performed at the same mall kiosk ▪ Patients had different earrings and different workers perform the piercings – Pus samples from all 3 patients revealed Pseudomonas aeruginosa infection – Oral antibiotics were prescribed but due to the lack of blood flow in cartilage, each patient needed surgical removal of the infected tissue Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (2 of 6) Summary of the case: – NP contacted the local health department to report her concerns about the piercing kiosk – Health officials investigated – Safety procedures at the kiosk dictated that a worker should: ▪ Decontaminate hands with an alcohol-based hand sanitizer ▪ Put on gloves ▪ Clean area of the ear to be pierced with an antiseptic-soaked cotton ball ▪ Slide the earring into the back of a manual tool ▪ Tool adjustments were made with gloved hands Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (3 of 6) Summary of the case: – Each worker claimed to follow safety guidelines – Health authorities sampled various surfaces and items at the kiosk – Antiseptic agent used to clean customer ears was half-empty and had been opened at least a month earlier Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (4 of 6) 1. What items and/or surfaces in the kiosk were probably sampled and why were they selected for sampling? 2. The kiosk workers claimed they couldn’t have transferred the pathogen from their hands to the customers because they wore gloves. Is this a valid conclusion? What are other ways P. aeruginosa could have been transferred to the customers during their piercing experience? Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (5 of 6) 3. Initially, the NP was concerned that the causative agent could have been Staphylococcus aureus, a Gram-positive bacterium, or P. aeruginosa, a Gram-negative bacterium; both are common culprits in piercing infections. What culture methods would allow for isolation and differentiation of these two bacteria? Would an anaerobic culture condition be needed? Be sure to explain your reasoning for all answers. 4. What role (if any) could the antiseptic have played in pathogen transmission? Copyright © 2023 Pearson Education, Inc. All Rights Reserved Think Clinically: Be S.M.A.R.T. About Cases (6 of 6) 5. Which tier would the piercing tool be classified for decontamination purposes? What precautions/protocols would the health authorities likely have recommended to limit future infections from piercings at the kiosk? 6. What is the most likely explanation for why the patient who had their lower earlobe pierced six months ago did not develop a P. aeruginosa infection? Copyright © 2023 Pearson Education, Inc. All Rights Reserved Copyright This work is protected by United States copyright laws and is provided solely for the use of instructors in teaching their courses and assessing student learning. Dissemination or sale of any part of this work (including on the World Wide Web) will destroy the integrity of the work and is not permitted. The work and materials from it should never be made available to students except by instructors using the accompanying text in their classes. All recipients of this work are expected to abide by these restrictions and to honor the intended pedagogical purposes and the needs of other instructors who rely on these materials. Copyright © 2023 Pearson Education, Inc. All Rights Reserved