Bacterial Staining and Nutrition PDF

BACTERIAL STAINING MS. REGINA MARIE TUQUIB Objectives of Staining  To determine the morphology of bacteria  To differentiate groups of bacteria  To identify organisms with special structures  Kinds of Ionizable Dyes Used in Staining Bacteria 1. Basic Dyes  These are cationic dyes...

BACTERIAL STAINING MS. REGINA MARIE TUQUIB Objectives of Staining  To determine the morphology of bacteria  To differentiate groups of bacteria  To identify organisms with special structures  Kinds of Ionizable Dyes Used in Staining Bacteria 1. Basic Dyes  These are cationic dyes with positively charged groups (pentavalent nitrogen) that adhere to negatively charged molecules like nucleic acids and proteins.  Examples are: Methylene blue, basic fuchsin, crystal violet, safranin, and malachite green  Kinds of Ionizable Dyes Used in Staining Bacteria 2. Acidic Dyes  These are anionic dyes with negatively charged groups that bind to positively charged cell structures  Examples are: Eosin, Rose Bengal and Acid Fuchsin  Staining Techniques Simple Techniques A single stain is used.  Itis directed towards coloring the forms and shapes all of the cells.  Example is the use of methylene blue.  Staining Techniques Differential Staining  It divides bacteria into separate groups.  It is directed towards coloring the components of the elements present.  Examples are Gram Staining and Acid Fast Bacilli Staining 1. Application of the primary stain. 2. Application of the mordant. 3. Application of the decolorizing agent. 4. Application of the secondary stain/counterstain  Staining Techniques Negative Staining  It is utilized to demonstrate the presence of diffuse capsule surrounding some bacteria.  It is an excellent technique for studying bacterial gas vacuoles and viral morphology.  It results in the bacteria appearing as light-colored bodies against background since the cell surface repels the acidic stain as a result of bacterial cells being negatively charged.  An example of this staining technique is the use of India ink or Nigrosin dye.  Staining Techniques Gram Stain  It is the most commonly used differential stain in the clinical microbiology laboratory.  It utilizes crystal violet as the primary stain while safranin is the secondary stain or counterstain.  In this staining procedure, iodine acts as the mordant while acetone-alcohol mixture acts as the decolorizing agent.  The "differential” process in Gram staining is the decolorization step because of the distinct appearance of bacteria after the application of the acetone-alcohol mixture.  Principle of Gram Staining  Bacteriawith thick cell walls containing teichoic acid retain the crystal violet-iodine complex dye after decolorization and appear purple, which means that they are Gram-positive.  Other bacteria with thinner cell walls that contain lipopolysaccharides do not retain the dye complex and appear deep pink or red, which means that they are Gram-negative.  Precautions during Gram staining 1. If the crystal violet dye is rinsed too vigorously prior to the application of iodine it will not be retained and will leave the Gram- negative bacteria unstained. 2. If the decolorization is prolonged, the Gram-positive complex will be removed and the Gram-positive bacteria will not be stained. 3. If the decolorization is insufficient, the organism may falsely appear as Gram-positive cells. General Rule of Gram staining All cocci are Gram-positive except for Neisseria, Veilonella, and Branhamella (Moraxella) General Rule of Gram staining All bacilli are Gram-negative except for Actinomadura, Arcanobacterium, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Gordonia, Kuthria, Listeria, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, Tropheryma whipplei, and Tsukamurella.  Reasons why Gram-Positive Bacteria Become Gram-Negative Bacteria 1. Aged, dying, and autolyzing cells Old cells may lose their ability to retain stains. Antibiotic-treated bacterial cells have atypical staining reaction. 2. By using acidic iodine during staining 3. Due to a technical error or the wrong use of stains Exceptions in Gram Staining: 1. Organisms that exist almost exclusively within host cells (Chlamydia) 2. Organisms that lack cell walls (Mycoplasma and Ureaplasma) 3. Organisms with insufficient dimension to be resolved by light microscopy (Spirochetes)  Staining Techniques Acid-Fast Staining  It is used to stain bacteria that have high lipid contents in their cell walls.  It utilizes carbol fuchsin as the primary stain and methylene blue or malachite green as the secondary stain.  Staining Techniques Acid-Fast Staining  In this procedure, the cell wall of acid-fast bacteria resists the acid-alcohol (hydrochloric acid-ethanol mixture) decolorization step.  Heat is applied as a mordant in the Ziehl-Neelsen method while tergitol is used in the Kinyoun method.  Principles of Acid-Fast Straining  The primary stain binds to the mycolic acid in the cell walls of the acid-fast bacteria, like in mycobacteria, and is retained after decolorizing with acid alcohol.  Acid-fast bacilli (AFB) retain the primary stain and deep- pink or red-colored while non-AFB are either blue- or green-colored (methylene blue or malachite green counterstains).  Acid-Fast Staining Methods 1. Ziehl-Neelsen/Hot staining method 2. Kinyoun's/Cold staining method 3. Pappenheim method - differentiates Mycobacterium smegmatis from Mycobacterium tuberculosis. 4. Baumgarten method - differentiates Mycobacterium leprae from Mycobacterium tuberculosis. 5. Auramine-rhodamine method - is selective for the cell wall of AFB.  Ways to Facilitate Acid-Fast Straining 1. The use of heating or steaming process for five to seven minutes to temporarily remove the mycolic acid, while the smear is flooded with stain 2. By increasing the concentration of dye and phenol in the staining reagent 3. A prolonged contact of the specimen with the primary stain 4. The addition of a wetting agent like tergitol  Modified Acid-Fast Staining method It is useful for the identification of intestinal coccidian oocysts.  Itis ideal for cryptosporidia and cyclospora parasites in specimens.  The specimens used are stools  Modified Acid-Fast Staining method  The reagents used are the same with those of the conventional acid-fast reagents except for the concentration of the acid alcohol (1% H,SO4).  The results show oocysts appearing as magenta- stained organisms against a blue background. Notes to Remember:  Acid-fastness is affected by colonial age, medium for growth, and UV light  The Ziehl-Neelsen method is ideal for concentrated smears and partially acid-fast bacilli like the Nocardia species.  Acid-alcohol decolorizing agent is composed of hydrochloric acid and ethanol. MICROBIAL NUTRITION MS. REGINA MARIE TUQUIB  Overview Microorganisms require specific nutrients that are essential for their growth and multiplication. Elements such as oxygen, carbon, hydrogen, nitrogen, and sulfur are some of the substances needed in energy production and biosynthesis. Further, temperature, pH, and moisture are also necessary to facilitate their growth and survival.  Physiologic Requirements of Bacteria According to Oxygen Requirement 1. AEROBES  These organisms require oxygen and grow well with room air.  Air contains 15% to 21% oxygen and 1% CO₂.  Some examples include Bordetella, Brucella, Mycobacteria and Pseudomonas.  Physiologic Requirements of Bacteria According to Oxygen a. Obligate anaerobes Requirement  These organisms absolutely do not require 2. ANAEROBES the presence of oxygen because they die after  These organisms do not prolonged exposure to air. require oxygen to grow.  Some examples are  These organisms have three Clostridium and different types: Bacteroides.  Physiologic Requirements of Bacteria b. Facultative anaerobes  They are the most clinically significant bacteria.  These organisms grow either in the presence or absence of oxygen; hence they are considered as aerobes that can grow anaerobically.  These organisms do not require oxygen but grow better in the presence.  Examples are the Enterobacteriaceae.  Physiologic Requirements of Bacteria c. Aerotolerant anaerobes  These organisms can survive in the presence of oxygen but will be unable to perform metabolic processes unless situated in an anaerobic environment.  An example is the Propionibacterium acnes  Physiologic Requirements of Bacteria According to Oxygen Requirement 3. MICROAEROPHILE  Itis an organism that requires 2% to 10% oxygen for growth.  Some examples are Campylobacter and Treponema pallidum  Physiologic Requirements of Bacteria According to Carbon Dioxide Requirement  Capnophiles require an increased CO₂ (5% to 10% CO₂).  Some examples of capnophiles are Haemophilus influenzae, Neisseria gonorrhoeae, and Streptococcus pneumoniae.  Most aerobic and facultative aerobic bacteria need 0.03% CO₂  Physiologic Requirements of Bacteria According to Nutritional Requirement As to the carbon source As to the electron source  Autotrophs use CO₂ as the sole source of carbon.  Lithotrophs reduce inorganic molecules to  Heterotrophs use reduced, performed, be used in biosynthesis or energy organic molecules from other bacteria. conservation. As to the energy source  Organotrophs require organic substances  Phototrophs are organisms that use light as (CHO, lipids) for growth and their energy source. multiplication.  Chemotrophs are organisms that utilize the energy produced by organic or inorganic compounds oxidation.  Notes to Remember  Fastidious bacteria require additional substances such as vitamins, purines, pyrimidines, and hemoglobin for growth and survival.  Saprophytes require dead organic substance.  Parasites require organic substances from living tissues.  Physiologic Requirements of Bacteria According to Temperature Requirement The optimum temperature for most bacteria is 35°C to 37°C. Psychrophiles/Cryophiles  These organisms grow well at 0°C to a maximum of 20°C.  Some examples are Listeria monocytogenes and Yersinia enterocolitica.  Physiologic Requirements of Bacteria Mesophiles  These organisms grow between 20°C to 45°C.  These are the most commonly encountered pathogenic bacteria in the clinical laboratory. Thermophiles/Hyperthermophiles  These organism grow between 50°C to 125°C.  Some examples are Bacillus stearothermophilus, Sulfolobus, Pyrococcus, Pyrodictium, and Thermus aquaticus.  Physiologic Requirements of Bacteria Extremophiles  These are prokaryotes that are able to survive in unusual conditions like the absence of oxygen, increased temperatures, and living below the earth’s surface (Bacillus infernus).  Terminologies Thermal Death Time - is the lowest/minimum time required to kill organisms under constant temperature. Thermal Death Point - is the lowest temperature required to kill organisms in a constant time  Physiologic Requirements of Bacteria According to pH pH scale is a measure of the hydrogen ion concentration of an organism’s environment. There are three (3) different types of organisms according to pH tolerance: a. Acidophiles - grow between pH 0 and 5.5 (Sulfolubus, Picrophilus, Aconitium). b. Neutrophiles - grow between pH 5.5 and 8.0 (most clinically significant bacteria). c. Alkalophiles - grow between pH 8.5 and 11.5 (Bacillus alcalophilus, Natronobacterium).  Notes to Remember  Theoptimum pH for most pathogenic bacteria is from pH 6.5 to 7.5.  Diagnostic laboratory culture media for bacterial isolation are usually adjusted to a final pH between 7.0 and 7.5.  Physiologic Requirements of Bacteria According to Moisture According to Pressure  Barophiles are organisms that grow  Moisture is vital for bacterial rapidly in high-pressure environments (600 growth and susceptibility testing. to 1100 atm pressure). According to Salt Concentration  Some examples are Photobacterium, Shewanella, and Colwellia.  Halophiles require and grow in increased concentration of According to Growth Factors sodium chloride.  Growth factors are substances that required by fastidious bacteria for their  Some examples are growth and multiplication. Staphylococus aureus and  Some examples of growth factors are Listeria monocytogens. amino acids, purines, pyrimidines, and vitamins.