Lab 4: Acid Fast Stain and Bacterial Growth Factors
Lab 4: Acid Fast Stain and Microbial Growth Factors
Acid Fast Stain
The acid-fast stain is a differential staining technique used to detect the presence or absence of mycolic acids within the bacterial cell wall.
Mycolic Acids:
These are waxy substances composed of fatty acids and fatty alcohols.
They are primarily found in the cell walls of bacteria belonging to the genera Mycobacterium and Nocardia.
Mycolic acid acts as a virulence factor, significantly aiding in the pathogenesis of these bacteria.
It allows bacteria to survive within phagocytes, contributing to their resistance against disinfectants and various antimicrobial therapies.
The waxy nature of the cell wall causes these bacteria to clump together, making Mycobacterium infections particularly challenging to treat.
Bacterial Cell Wall Comparison:
Unlike Gram-positive and Gram-negative bacteria, which primarily feature peptidoglycan layers (and an outer membrane in Gram-negatives), Mycobacteria possess a unique cell wall structure.
Their cell wall includes mycolic acid, glycolipid, galactan, LAM (Lipoarabinomannan), and mannophosphoinositide layers, in addition to peptidoglycan.
Clinical Significance: Presumptive Test for Mycobacterium and Nocardia
Purpose: This stain is a crucial presumptive test for suspected infections involving Mycobacterium or Nocardia.
Mycobacterium tuberculosis and Tuberculosis (TB):
Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease that historically infects approximately one-third of the global human population.
Re-emerging Infection: TB is currently considered a re-emerging infection, meaning it has reappeared as a significant health concern worldwide after a period of decline.
Other examples of re-emerging diseases include malaria, cholera, pertussis, influenza, pneumococcal disease, and gonorrhea.
Challenges: Mycobacterium tuberculosis's re-emergence is largely due to the development of drug resistance to multiple antibiotics.
Treatment: Effective treatment typically involves a combination of antibiotics taken for a prolonged period, often ranging from 6 to 12 months.
Relevance: TB remains a critical consideration for those entering healthcare professions due to its global impact and evolving treatment challenges.
Global Health Perspective:
A 2020 NYTimes article described TB as "The Biggest Monster," responsible for 1.5 million deaths annually and 10 million cases per year, suggesting its toll was not yet comparable to COVID-19 at that time.
Case Studies/Outbreaks in Las Vegas:
2013 Neonatal Ward Alert: 26 people were infected after a mother and her newborn twin girls died from TB. The mother was believed to have contracted a rare strain from unpasteurized dairy products overseas.
2017 Teacher's Death: A Las Vegas teacher died from TB, leading to the screening of over 100 students and healthcare workers. Of those screened, 4 tested positive for latent tuberculosis (non-infectious) and none for active tuberculosis.
2023 School Outbreak: An individual with active TB, symptomatic for over a year before diagnosis, visited more than 26 school campuses. Health officials investigated potential exposures at 18 schools and a training center, issuing notifications.
Active TB causes symptoms like coughing, fever, and chills, and is infectious.
Latent TB infection shows no symptoms, is not infectious, and can be treated to prevent active disease.
TB in the United States:
After nearly 3 decades of consistent decline and a significant drop in 2020, TB cases and rates began increasing in 2021. This trend continued into 2024.
Provisional CDC data for 2024 reported 10,347 TB cases, with a rate of 3.0 cases per 100,000 population.
The percentage increase from 2023 to 2024 was 8\% for case counts and 6\% for rates, which moderated from the 15\% increases seen from 2022 to 2023.
Factors contributing to this rise include recovery from pandemic-related healthcare disruptions, increased post-pandemic travel and migration, and outbreaks in several states.
Acid Fast Stain Reagents and Procedure (Kinyoun Method)
Principle: The presence of mycolic acid prevents successful staining by most routine methods.
Kinyoun Method (Cold Acid-Fast Stain):
Primary Dye: Carbolfuchsin
A phenolic compound and lipid-soluble primary stain.
Contains phenol, which helps the stain penetrate the waxy mycolic acid layer of the cell wall.
Forms a fuchsia-pink complex within acid-fast cells.
Mycolic acids impart a high affinity for carbolfuchsin and resist decolorization with acid alcohol, a characteristic known as acid-fastness.
Decolorizer: Acid Alcohol
Designed to strip the carbolfuchsin stain from all non-acid-fast cells.
It does not penetrate the waxy cell wall of acid-fast organisms, thus they retain the primary stain.
Counterstain: Methylene Blue
Stains bacteria that are not acid-fast (because they were decolorized) to appear blue under the microscope.
Results Interpretation:
Acid-fast positive: Appear fuchsia-pink (e.g., Mycobacterium smegmatis, Mycobacterium tuberculosis).
Acid-fast negative: Appear blue (e.g., Staphylococcus aureus, Bacillus cereus).
Common Mistakes in Acid Fast Staining
Sticky Mycobacteria: Mycobacteria are notorious for their stickiness, making them difficult to transfer from a loop.
Solution: Mix the sample into the pool of bacteria on the slide for longer than a second, ideally around 5 seconds, to ensure fair distribution of organisms.
Incorrect Decolorizer: Do not use Gram's alcohol (used for Gram staining) instead of acid alcohol.
Consequence: If Gram's alcohol is used, everything will stain acid-fast positive.
Decolorization Time: This is the most critical step.
Decolorize for approximately 5-30 seconds ( hicksim10 drops).
Over-decolorization can occur if too much alcohol is added, leading to false-negative results.
Aseptic Technique: Ensure proper aseptic technique throughout the procedure.
Bacteria should not be mixed until they are on the slide.
Growth Factors: Temperature
Overview: Bacteria exhibit a remarkable ability to grow across a wide range of temperatures, from below 0^ ext{o}C to over 110^ ext{o}C. Their cell structures, proteins, and enzymes are uniquely adapted to function within these specific thermal environments.
Key Temperature Definitions:
Optimum Temperature: The ideal temperature at which an organism demonstrates its best growth.
Minimum Temperature: The lowest temperature at which an organism can sustain growth.
Maximum Temperature: The highest temperature at which an organism can sustain growth.
Measuring Growth:
Optimal growth in broth (liquid) cultures is typically observed and quantified by measuring turbidity (cloudiness).
Turbidity is measured using a spectrophotometer, which passes a beam of light through the sample and measures the absorbance of light.
There is a proportional relationship: higher absorbance of light indicates higher growth, as more cells scatter light. The absorbance reading is also known as optical density.
Microorganism Classification by Temperature:
Psychrophiles: Grow exclusively below 20^ ext{o}C (e.g., found in Arctic and Antarctic regions).
Psychrotrophs: Can function at cold temperatures but are capable of surviving up to 35^ ext{o}C.
Examples include bacteria found on chilled meat, contributing to product spoilage (e.g., Pseudomonas spp., which spoil fruits, vegetables, grains, milk, meat, poultry, eggs, fish, and processed foods).
They can even survive pasteurization.
Mesophiles: Adapted to moderate temperatures, typically between 15^ ext{o}C and 45^ ext{o}C (e.g., human body temperature is around 37^ ext{o}C).
Most bacterial infections in humans are caused by mesophiles.
Beneficial bacteria in human intestinal flora (Lactobacillus acidophilus) are also mesophiles.
Examples include Listeria monocytogenes, Pseudomonas maltophilia, Staphylococcus aureus, Streptococcus pyrogenes, E. coli, and Clostridium kluyveri.
Thermophiles: Grow optimally at temperatures ranging from 45^ ext{o}C to 80^ ext{o}C (e.g., found in hot springs).
Extreme Thermophiles: Capable of surviving at very high temperatures, from 80^ ext{o}C to 110^ ext{o}C (e.g., found in deep ocean thermal vents).
Thermoduric: Generally mesophilic organisms that can withstand high temperatures (70^ ext{o}C or higher) for short periods.
Examples include heat-resistant bacteria in milk (Bacillus, Clostridium, and Enterococci) that survive pasteurization, impacting milk product quality.
Temperature Procedure:
Materials: Two sets of four broth tubes.
Labeling: One set labeled E. coli for temperatures: 4^ ext{o}C, 25^ ext{o}C, 36^ ext{o}C, 55^ ext{o}C. The other set labeled B. stearothermophilus for the same temperatures.
Inoculation: Inoculate each E. coli tube with 10 ext{ ul} of E. coli, and each B. stearothermophilus tube with 10 ext{ ul} of B. stearothermophilus, using a 2-20 ext{ ul} pipette and aseptic technique.
Incubation: Place inoculated tubes into their respective temperature racks.
Hypothesis: Students are required to develop hypotheses about anticipated growth results for each temperature.
Growth Factors: Water Activity and Osmotic Pressure
Importance of Water: Water is essential for life, maintaining turgor pressure within bacterial cells, regulating pH, and supporting metabolic processes.
Osmosis: Bacteria maintain high cytoplasmic solute concentrations, which drives the inward diffusion of water into the cell via osmosis.
Osmosis is the diffusion of water across a semi-permeable membrane, occurring due to differences in solute concentrations on either side of the membrane. It aims to equalize solute concentrations.
Types of Osmotic Environments:
Isotonic Environments: Solute concentrations are equal on both sides of the membrane. There is no net movement of water in either direction.
Hypotonic Environments: The surrounding environment has a lower solute concentration than the cell cytoplasm.
Water moves into the cell, causing it to swell and increasing turgor pressure.
Bacterial cells are protected from bursting by their rigid cell walls.
Hypertonic Environments: The surrounding environment has a higher solute concentration than the cell cytoplasm.
Water leaves the cell, moving into the surrounding environment.
The cell membrane shrinks away from the cell wall, a process known as plasmolysis.
Microorganism Classification by Osmolarity:
Halophiles: Microbes that can only live and grow in high salinity (hypertonic) environments, typically around 5-15\%. Most are Archaea, but some are bacteria.
Moderate Halophile: Grow in 4.7-20\% salinity.
Extreme Halophiles: Can only grow in extremely saline environments (20-30\%), such as the Dead Sea or the Great Salt Lake.
Halotolerant: Microbes that can survive in a range of salinity (from none to slightly higher) and grow in environments of 0-11\% salinity.
Osmophiles: Microbes that can tolerate very high sugar concentrations, and thus high osmotic pressures.
Water Activity vs. Salinity: Water activity is determined by the total solute concentration, which can include not only salt but also sugars, amino acids, or other molecules.
Water Activity and Osmotic Pressure Procedure:
Materials: Six salt agar plates with varying NaCl concentrations (labeled 0\%, 5\%, 10\%, 15\%, 20\%, and 25\%).
Inoculation: Spot 10 ext{ µl} of samples onto each plate. Do not spread the fluid.
Incubation: Invert the plates once the fluid is absorbed, label them with group initials, and incubate at 37^ ext{o}C.
Growth Factors: Aerotolerance
Significance: Oxygen requirements are crucial for culturing and identifying microbes, as oxygen concentrations vary widely in natural environments (e.g., soil) and within animal bodies (e.g., the gastrointestinal tract is largely anaerobic).
Oxygen and Metabolism:
While oxygen is beneficial for producing ATP through aerobic cellular respiration, this process also generates toxic byproducts known as reactive oxygen species (ROS).
ROS can damage vital cellular components, such as DNA.
Microbes must possess specific enzymes to inactivate ROS. Those lacking these enzymes cannot tolerate oxygen.
Alternatively, microbes without ROS-inactivating enzymes utilize fermentation or anaerobic respiration (employing other terminal electron acceptors like sulfate or nitrate) to produce ATP.
Microorganism Classification by Oxygen Requirements:
Strict (Obligate) Aerobes: Microbes that are entirely dependent on oxygen as the final electron acceptor for cellular respiration to produce ATP. They cannot survive in the absence of oxygen.
Facultative Anaerobes: Microbes capable of using oxygen but do not strictly require it. They can grow in both the presence and absence of oxygen.
They possess both respiratory and fermentative metabolism, often featuring a branched electron transport chain that allows them to use multiple electron acceptors (e.g., oxygen, nitrate, sulfate).
Aerobic respiration is more efficient, so facultative anaerobes typically grow better with oxygen.
Strict (Obligate) Anaerobes: Microbes that cannot grow at all in the presence of oxygen. They rely on fermentation and/or anaerobic respiration for ATP synthesis and are killed by oxygen.
Aerotolerant Anaerobes: Microbes that can tolerate the presence of oxygen but do not utilize it to produce ATP. Unlike strict anaerobes, they are not harmed by oxygen.
Microaerophiles: Microbes that require oxygen for growth but can only tolerate low concentrations of oxygen.
Aerotolerance Procedure:
Materials: Two TSA (Tryptic Soy Agar) plates and unknown agar cultures labeled A, B, and C.
Labeling: Divide each TSA plate into thirds, labeling each third A, B, and C. Label one plate 'O2' and the other 'No O2', adding group initials.
Inoculation: Use a loop and aseptic technique to inoculate a single streak of each unknown bacterium (A, B, C) in its designated third on both plates.
Incubation: Invert the 'O2' plate and place it on a tray for incubation in an oxygen-rich environment. Place the 'No O2' plate into an anaerobic bag for anaerobic growth.
General Laboratory Reminders
Gram Staining: Practice and review Gram staining techniques.
Streak for Isolation:
Examine your streak for isolation plates for well-isolated colonies.
A well-isolated colony should have a clear area around it, roughly equal to the width of the inoculating loop, in all directions.
If well-isolated colonies are not achieved, seek feedback from the instructor and repeat the streak for isolation on a new TSA plate (this will be the final opportunity to perform this before the practical exam).
Culture Media Classifications:
General Purpose Media: Supports the growth of a wide range of microorganisms that do not require specialized growth factors (e.g., Nutrient broth, Tryptic Soy Broth, Tryptic Soy Agar (TSA)).
Enrichment Media: Contains specific growth factors required by certain fastidious microorganisms (e.g., Blood Agar Plate (BAP), which includes sheep red blood cells. Some microbes lyse these cells to obtain nutrients like heme).
Selective Media: Inhibits the growth of some groups of microorganisms while promoting the growth of others (e.g., Sabouraud's Dextrose Agar (SDA), with its high dextrose concentration, inhibits most bacteria but encourages fungal (mold and yeast) growth).
Differential Media: Allows for differentiation between microbial colonies based on their distinct cultural characteristics (e.g., Blood Agar is also differential, as different patterns of hemolysis (lysis of blood cells) can be observed and are characteristic for certain bacteria).
Laboratory Housekeeping
Tablets: Students must sign out of tablets and then shut them down.
Incinerators: Ensure all incinerators are turned off.
Waste Disposal: Dispose of waste properly.
No gloves in regular trash.
Gram staining waste must be disposed of in biohazard containers.
Microscopes: Crucially, check microscopes for oil! There have been persistent complaints about oil on objectives, focus knobs, and stages across campuses. Also, ensure no slides are left on the microscopes and that Gram trays are cleaned after use.