This document describes bacterial staining techniques including Gram staining and acid-fast staining. Gram staining differentiates bacteria based on cell wall structure and stains Gram-positive bacteria purple and Gram-negative bacteria pink or red. Acid-fast staining identifies acid-fast bacteria that retain the primary stain carbolfuchsin despite decolorization. Procedures for both Gram staining and acid-fast staining of mixed bacterial samples are provided along with questions about the techniques.
Enumeration is counting of microorganisms present in a sample.
This is done to know the intense of presence of the spoilers in the spoiled food.
To detect which type of organism is responsible for the spoilage.
Mostly this is done two important methods.
Viable count
Total count
VIABLE COUNT:
A viable cell count allows one to identify the number of actively growing or dividing cells in a sample.
The plate count method or spread plate method relies on bacteria growing a colony on a nutrient medium.
Number of colonies can be counted.
Plate count agar is used for general count
MacConkey agar is used for Gram negative organisms.
TOTAL COUNT:
The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count. The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count.
Pour plate method:
The same procedure is done for this till serial dilution.
The serially diluted sample is then mixed with the molten nutrient agar.
Then poured onto the sterile petridish.
Incubated under appropriate temperature amd the colonies where counted.
ConclusionThe enumeration of these spoiled food samples are important to encounter the type of microbe is causing the spoilage.
And hence this is used to prevent the same type of spoilage.
This can be avoided by making the environmental changes which inhibits the organism which is responsible for the spoilage.
The document discusses the management and use of laminar air flow for explant inoculation in tissue culture. It describes laminar air flow as an enclosed bench designed to provide sterile conditions through HEPA filtration and UV sterilization of air. Proper management includes sterilizing surfaces with ethanol before and after use, turning on UV lights 30 minutes prior, and flame sterilizing instruments. Explants are surface sterilized with mercuric chloride before trimming and inoculating into sterile culture media in the laminar flow hood. Inoculated cultures are then transferred to a sterilized culture room. Safety precautions for laminar flow use include switching off UV lights during use, cleaning hands with ethanol, and properly adjusting
This document discusses laminar air flow hoods, including their purpose of providing sterile work environments, types (horizontal and vertical flow), working principles involving HEPA filtration of air, proper use and maintenance, and validation processes to ensure sterility through design, installation, operational, and performance qualifications. Laminar flow hoods use HEPA-filtered air flowing in a laminar pattern to maintain aseptic conditions for activities like cell culture work by removing airborne contaminants.
This document describes the Limulus Amoebocyte Lysate (LAL) test used to detect bacterial endotoxins. It involves mixing the LAL reagent, obtained from horseshoe crab blood, with test samples. If endotoxins are present, the LAL reagent will clot, forming a gel. The procedure involves incubating mixtures of the LAL reagent with test samples, positive controls containing endotoxins, and negative controls. After incubation, the mixtures are observed to see if gels formed, indicating the presence of endotoxins above permissible limits. The LAL test is a common method used in the pharmaceutical industry to test for bacterial endotoxins in products.
Sterility testing is performed on pharmaceutical products to detect any viable microorganisms that could contaminate the products. There are two main methods for sterility testing - membrane filtration and direct inoculation. Membrane filtration involves filtering a sample through a membrane and incubating portions of the membrane in culture media to detect any microbes. Direct inoculation adds a sample directly to culture media and incubates it. Products like injections, implants, and dressings undergo sterility testing to assure their safety before use.
Gram staining is used to classify bacteria based on differences in cell wall composition. It involves staining with crystal violet and iodine, then decolorizing and counterstaining. Gram-positive bacteria retain the crystal violet stain while gram-negative bacteria take up the counterstain. Acid-fast staining is used to identify mycobacteria by using heat to bind primary stain within acid-fast cell walls. Capsule staining uses India ink's dark background to contrast unstained capsules around stained cells. Spore staining employs malachite green and heat to permanently stain endospores within decolorized vegetative cells.
Microbial limit tests I.P by Dr.P.srinivas cnupogu
The document describes microbial limit testing methods for pharmaceutical and cosmetic materials. There are two main methods: testing for specific pathogenic microorganisms like E. coli, Salmonella species, S. aureus, and P. aeruginosa and determining the total aerobic microbial count. The total aerobic microbial count method involves pre-treating samples depending on solubility, filtering through a membrane, incubating, and calculating microorganisms per unit weight or volume. Confirmation tests are described to detect specific microorganisms using selective agar media, biochemical tests, and colony characteristics.
General analytical methods of milk powder finalSkAzizuddin1
This document outlines various analytical methods for dried milk powder, including procedures for determining moisture, fat, acidity, carbohydrates, and detecting adulterants. Moisture is determined by drying a sample to constant weight. Fat is extracted using solvents and weighed. Acidity is measured by titration. Carbohydrates are calculated by subtracting moisture, fat, and other components from 100%. Adulterants can be detected using tests for cane sugar, urea, preservatives, and neutralizers.
Enumeration is counting of microorganisms present in a sample.
This is done to know the intense of presence of the spoilers in the spoiled food.
To detect which type of organism is responsible for the spoilage.
Mostly this is done two important methods.
Viable count
Total count
VIABLE COUNT:
A viable cell count allows one to identify the number of actively growing or dividing cells in a sample.
The plate count method or spread plate method relies on bacteria growing a colony on a nutrient medium.
Number of colonies can be counted.
Plate count agar is used for general count
MacConkey agar is used for Gram negative organisms.
TOTAL COUNT:
The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count. The initial analysis is done by mixing serial dilution of sample in liquid nutrient agar which is then poured into bottles.
The bottles are then sealed and laid on their sides to produce a slopping agar surface.
The colonies are then counted by eye.The total number of colonies are said as Total Viable Count.
Pour plate method:
The same procedure is done for this till serial dilution.
The serially diluted sample is then mixed with the molten nutrient agar.
Then poured onto the sterile petridish.
Incubated under appropriate temperature amd the colonies where counted.
ConclusionThe enumeration of these spoiled food samples are important to encounter the type of microbe is causing the spoilage.
And hence this is used to prevent the same type of spoilage.
This can be avoided by making the environmental changes which inhibits the organism which is responsible for the spoilage.
The document discusses the management and use of laminar air flow for explant inoculation in tissue culture. It describes laminar air flow as an enclosed bench designed to provide sterile conditions through HEPA filtration and UV sterilization of air. Proper management includes sterilizing surfaces with ethanol before and after use, turning on UV lights 30 minutes prior, and flame sterilizing instruments. Explants are surface sterilized with mercuric chloride before trimming and inoculating into sterile culture media in the laminar flow hood. Inoculated cultures are then transferred to a sterilized culture room. Safety precautions for laminar flow use include switching off UV lights during use, cleaning hands with ethanol, and properly adjusting
This document discusses laminar air flow hoods, including their purpose of providing sterile work environments, types (horizontal and vertical flow), working principles involving HEPA filtration of air, proper use and maintenance, and validation processes to ensure sterility through design, installation, operational, and performance qualifications. Laminar flow hoods use HEPA-filtered air flowing in a laminar pattern to maintain aseptic conditions for activities like cell culture work by removing airborne contaminants.
This document describes the Limulus Amoebocyte Lysate (LAL) test used to detect bacterial endotoxins. It involves mixing the LAL reagent, obtained from horseshoe crab blood, with test samples. If endotoxins are present, the LAL reagent will clot, forming a gel. The procedure involves incubating mixtures of the LAL reagent with test samples, positive controls containing endotoxins, and negative controls. After incubation, the mixtures are observed to see if gels formed, indicating the presence of endotoxins above permissible limits. The LAL test is a common method used in the pharmaceutical industry to test for bacterial endotoxins in products.
Sterility testing is performed on pharmaceutical products to detect any viable microorganisms that could contaminate the products. There are two main methods for sterility testing - membrane filtration and direct inoculation. Membrane filtration involves filtering a sample through a membrane and incubating portions of the membrane in culture media to detect any microbes. Direct inoculation adds a sample directly to culture media and incubates it. Products like injections, implants, and dressings undergo sterility testing to assure their safety before use.
Gram staining is used to classify bacteria based on differences in cell wall composition. It involves staining with crystal violet and iodine, then decolorizing and counterstaining. Gram-positive bacteria retain the crystal violet stain while gram-negative bacteria take up the counterstain. Acid-fast staining is used to identify mycobacteria by using heat to bind primary stain within acid-fast cell walls. Capsule staining uses India ink's dark background to contrast unstained capsules around stained cells. Spore staining employs malachite green and heat to permanently stain endospores within decolorized vegetative cells.
Microbial limit tests I.P by Dr.P.srinivas cnupogu
The document describes microbial limit testing methods for pharmaceutical and cosmetic materials. There are two main methods: testing for specific pathogenic microorganisms like E. coli, Salmonella species, S. aureus, and P. aeruginosa and determining the total aerobic microbial count. The total aerobic microbial count method involves pre-treating samples depending on solubility, filtering through a membrane, incubating, and calculating microorganisms per unit weight or volume. Confirmation tests are described to detect specific microorganisms using selective agar media, biochemical tests, and colony characteristics.
General analytical methods of milk powder finalSkAzizuddin1
This document outlines various analytical methods for dried milk powder, including procedures for determining moisture, fat, acidity, carbohydrates, and detecting adulterants. Moisture is determined by drying a sample to constant weight. Fat is extracted using solvents and weighed. Acidity is measured by titration. Carbohydrates are calculated by subtracting moisture, fat, and other components from 100%. Adulterants can be detected using tests for cane sugar, urea, preservatives, and neutralizers.
This document discusses sterility testing methods according to various pharmacopoeias. It provides details on membrane filtration and direct inoculation methods for testing sterility of pharmaceutical products like injections and ophthalmic preparations. These methods are based on incubating the product samples in fluid thioglycollate medium and soybean-casein digest medium to check for microbial growth. Validation of sterility testing methods and interpretation of results are also covered.
A Haemocytometer is a specialized and calibrated microscope slide that has a counting chamber with a known volume of liquid, which designed to allow operators to quickly estimate the concentration of cells in a sample.
McFarland standards are used in microbiology to standardize the turbidity of bacterial suspensions for consistent microbial testing. They involve mixing barium chloride and sulfuric acid to form a barium sulfate precipitate, causing turbidity. A 0.5 McFarland standard is prepared by mixing specific amounts of barium chloride and sulfuric acid. Now, latex particles are used instead to increase the shelf life of the McFarland standards, which are compared to bacterial suspensions to adjust their turbidity.
The document discusses the disk diffusion method for testing bacterial susceptibility to antimicrobial drugs. Key points:
1. The disk diffusion method involves placing disks containing known amounts of antimicrobial drugs onto agar plates inoculated with bacteria. As the drugs diffuse out, a gradient is established and zones of inhibition form where growth is inhibited.
2. The size of the zones of inhibition depends on factors like the drug quantity, bacteria susceptibility, agar depth and bacteria concentration.
3. By measuring zone sizes and comparing to standards, bacteria can be classified as susceptible, intermediate or resistant to each drug tested. This helps determine appropriate antibiotic treatment.
Managing and maintaining the integrity of laboratories is challenging, particularly if contamination is discovered. During this webinar, you will learn about several decontamination and disinfection techniques used to decontaminate spaces and equipment.
Structure and reproduction of Aspergillus niger ,with picture of different reproduction methods in detail ,also called sac fungi,large groupof true fungi ,saprophyte,it also known to cause food contaminations or food spoilage ,also cause black mold in fruits and vegetables like grapes, apricote ,onions and peanuts .Aspergillus niger is common group of Aspergillus.reproduction by sexual ,asexual or vegetative methods. vegetative mthods by fragmentation ,sclerotia
This document discusses different types of incubators and their applications. It begins by describing laboratory incubators, which maintain optimal temperature, humidity, and gases to grow cell and microbiological cultures. The market is divided into gassed CO2 incubators and non-gassed types. Incubators provide controlled environments for cultures and protect cells from temperature and atmospheric changes. Types discussed include standard, cooled, humidity-controlled, CO2, shaking, and hybridization incubators. Factors like volume, materials, temperature control, and safety features are considered when choosing an incubator. Applications include growing cell cultures, microbiological analyses, breeding insects, storing samples, and growing protein crystals.
This document discusses several culture techniques used to diagnose nematode infections based on larval morphology:
1. The Harada-Mori filter paper culture uses a filter paper strip coated with feces and placed in a test tube with water to allow hatching and development of hookworm, Strongyloides, and Trichostrongylus larvae over 10 days.
2. The Baermann technique uses a funnel with gauze and feces placed over a water reservoir, allowing rhabditiform and filariform Strongyloides and hookworm larvae to migrate through the gauze into collected fluid over 2-12 hours.
3. Charcoal culture and filter paper/slant culture
The document discusses various methods for preserving bacterial and fungal strains. It describes preservation techniques such as serial transfer, preservation in distilled water, under oil, lyophilization, on silica gel, paper, beads and soil. It also discusses cryopreservation techniques like storing agar plugs or cell suspensions in liquid nitrogen. The goals of preservation are to maintain culture productivity, genetic purity and biochemical properties over long periods of storage and transportation. The document provides detailed protocols for various preservation methods.
The document discusses different types of microtomes used to cut extremely thin slices of materials for examination under microscopes. It describes 7 types of microtomes: sledge microtomes cut thick sections up to 60 μm for light microscopy; rotary microtomes cut sections from 1-60 μm using a rotating knife; cryomicrotomes cut frozen samples in a liquid nitrogen chamber; ultramicrotomes cut extremely thin 40-100 nm sections for electron microscopy; vibrating microtomes cut sections of 30-500 μm for difficult biological samples using a vibrating blade; saw microtomes use a rotating saw to cut hard materials like teeth or bones in sections of about 30 μm; and laser microtomes use
This document discusses methods for isolating bacteria from mixed cultures in order to obtain a pure culture of a single bacterial species. It describes several techniques used for isolation including streaking, plating, dilution, enrichment procedures, and single cell techniques. Streaking is the most widely used method and involves streaking bacteria across an agar plate with a sterile loop or needle to separate individual colonies. Other methods like plating, dilution, and enrichment procedures help isolate bacteria by taking advantage of differences in growth rates or nutritional requirements. Obtaining a pure culture of a single bacterial species is the first step in identifying bacteria that may cause disease.
Glassware apparatus and their uses(pdf)Science Equip
Glassware is commonly used in laboratories to contain and transport liquids. Beakers can hold fluids for mixing, heating, and reactions, though their volume measurements may be approximate. Funnels are used for pouring and filtration, with separatory funnels having features to prevent spillage. Pipets precisely measure fluid amounts, while volumetric flasks are used to create precise solutions by filling to an etched line. All glassware should be cleaned immediately after use.
Laboratory glassware is usually made of borosilicate glass, which is resistant to chemicals except hydrofluoric acid. Common types of glassware include beakers for holding solutions, flasks for heating liquids, volumetric flasks for precisely measuring volumes, and pipettes for dispensing precise amounts of liquids. Pipettes can be graduated or volumetric, and are used to transfer reagents and biological samples in clinical testing.
Microbial Culture Preservation and its MethodsDENNISMMONDAH1
This document discusses various methods for preserving microbial cultures, including short term and long term methods. Short term methods include periodic transfer to fresh media, preservation using saline suspension, drying, and refrigeration. Long term methods discussed are preservation using liquid paraffin/mineral oil, glycerol, lyophilization (freeze drying), and cryopreservation in liquid nitrogen. The aim of preservation is to maintain cultures in viable condition for extended periods without genetic changes.
This document provides an analysis of butter, including its definition, characteristics, apparatus used in analysis, and methods for determining its moisture, fat, curd, and salt content. Butter is defined as a smooth fatty food made from milk or cream that contains at least 80% milk fat. Its characteristics include a firm waxy body and clean cut when sliced. Various apparatus are used like crucibles, filters, flasks, and ovens. Methods described include preparing the butter sample, then determining moisture by heating and weighing. Fat is extracted using petroleum ether, and curd content is calculated. Salt content can be determined using Volhard's or Mohr's titration methods. Microbial contamination sources and controls are also outlined.
The document discusses various staining techniques used to visualize bacteria under a microscope. It describes the principles of simple staining using single dyes like methylene blue or carbol fuchsin. Differential staining techniques like Gram staining and acid-fast staining are also covered, which use multiple dyes to categorize bacteria based on cell wall characteristics. Special stains used to highlight specific bacterial structures such as capsules, flagella or spores are mentioned. Detailed procedures for common staining methods like Gram stain, acid-fast stain and Albert stain are provided. The document aims to explain the use of staining to differentiate bacterial types and visualize their morphology.
This document provides instructions for a laboratory exercise on using the compound microscope, including identifying microscope parts, proper care techniques, calculating magnification, and utilizing different lens powers. It explains that the compound microscope uses multiple lenses to magnify objects beyond what is visible to the naked eye. Proper use of immersion oil and understanding numerical aperture allow for gathering more light and achieving better image resolution under the microscope.
This laboratory report summarizes three experiments conducted by a group of students:
1. Media preparation demonstration - The group prepared two types of nutrient agar media.
2. Isolation of soil bacteria - The group determined the viable titer of soil bacteria by serial dilution and plating, then isolated pure cultures.
3. Staining techniques - The group demonstrated endospore staining of Bacillus and gram staining of an unknown bacterium to identify cell structure.
This document discusses sterility testing methods according to various pharmacopoeias. It provides details on membrane filtration and direct inoculation methods for testing sterility of pharmaceutical products like injections and ophthalmic preparations. These methods are based on incubating the product samples in fluid thioglycollate medium and soybean-casein digest medium to check for microbial growth. Validation of sterility testing methods and interpretation of results are also covered.
A Haemocytometer is a specialized and calibrated microscope slide that has a counting chamber with a known volume of liquid, which designed to allow operators to quickly estimate the concentration of cells in a sample.
McFarland standards are used in microbiology to standardize the turbidity of bacterial suspensions for consistent microbial testing. They involve mixing barium chloride and sulfuric acid to form a barium sulfate precipitate, causing turbidity. A 0.5 McFarland standard is prepared by mixing specific amounts of barium chloride and sulfuric acid. Now, latex particles are used instead to increase the shelf life of the McFarland standards, which are compared to bacterial suspensions to adjust their turbidity.
The document discusses the disk diffusion method for testing bacterial susceptibility to antimicrobial drugs. Key points:
1. The disk diffusion method involves placing disks containing known amounts of antimicrobial drugs onto agar plates inoculated with bacteria. As the drugs diffuse out, a gradient is established and zones of inhibition form where growth is inhibited.
2. The size of the zones of inhibition depends on factors like the drug quantity, bacteria susceptibility, agar depth and bacteria concentration.
3. By measuring zone sizes and comparing to standards, bacteria can be classified as susceptible, intermediate or resistant to each drug tested. This helps determine appropriate antibiotic treatment.
Managing and maintaining the integrity of laboratories is challenging, particularly if contamination is discovered. During this webinar, you will learn about several decontamination and disinfection techniques used to decontaminate spaces and equipment.
Structure and reproduction of Aspergillus niger ,with picture of different reproduction methods in detail ,also called sac fungi,large groupof true fungi ,saprophyte,it also known to cause food contaminations or food spoilage ,also cause black mold in fruits and vegetables like grapes, apricote ,onions and peanuts .Aspergillus niger is common group of Aspergillus.reproduction by sexual ,asexual or vegetative methods. vegetative mthods by fragmentation ,sclerotia
This document discusses different types of incubators and their applications. It begins by describing laboratory incubators, which maintain optimal temperature, humidity, and gases to grow cell and microbiological cultures. The market is divided into gassed CO2 incubators and non-gassed types. Incubators provide controlled environments for cultures and protect cells from temperature and atmospheric changes. Types discussed include standard, cooled, humidity-controlled, CO2, shaking, and hybridization incubators. Factors like volume, materials, temperature control, and safety features are considered when choosing an incubator. Applications include growing cell cultures, microbiological analyses, breeding insects, storing samples, and growing protein crystals.
This document discusses several culture techniques used to diagnose nematode infections based on larval morphology:
1. The Harada-Mori filter paper culture uses a filter paper strip coated with feces and placed in a test tube with water to allow hatching and development of hookworm, Strongyloides, and Trichostrongylus larvae over 10 days.
2. The Baermann technique uses a funnel with gauze and feces placed over a water reservoir, allowing rhabditiform and filariform Strongyloides and hookworm larvae to migrate through the gauze into collected fluid over 2-12 hours.
3. Charcoal culture and filter paper/slant culture
The document discusses various methods for preserving bacterial and fungal strains. It describes preservation techniques such as serial transfer, preservation in distilled water, under oil, lyophilization, on silica gel, paper, beads and soil. It also discusses cryopreservation techniques like storing agar plugs or cell suspensions in liquid nitrogen. The goals of preservation are to maintain culture productivity, genetic purity and biochemical properties over long periods of storage and transportation. The document provides detailed protocols for various preservation methods.
The document discusses different types of microtomes used to cut extremely thin slices of materials for examination under microscopes. It describes 7 types of microtomes: sledge microtomes cut thick sections up to 60 μm for light microscopy; rotary microtomes cut sections from 1-60 μm using a rotating knife; cryomicrotomes cut frozen samples in a liquid nitrogen chamber; ultramicrotomes cut extremely thin 40-100 nm sections for electron microscopy; vibrating microtomes cut sections of 30-500 μm for difficult biological samples using a vibrating blade; saw microtomes use a rotating saw to cut hard materials like teeth or bones in sections of about 30 μm; and laser microtomes use
This document discusses methods for isolating bacteria from mixed cultures in order to obtain a pure culture of a single bacterial species. It describes several techniques used for isolation including streaking, plating, dilution, enrichment procedures, and single cell techniques. Streaking is the most widely used method and involves streaking bacteria across an agar plate with a sterile loop or needle to separate individual colonies. Other methods like plating, dilution, and enrichment procedures help isolate bacteria by taking advantage of differences in growth rates or nutritional requirements. Obtaining a pure culture of a single bacterial species is the first step in identifying bacteria that may cause disease.
Glassware apparatus and their uses(pdf)Science Equip
Glassware is commonly used in laboratories to contain and transport liquids. Beakers can hold fluids for mixing, heating, and reactions, though their volume measurements may be approximate. Funnels are used for pouring and filtration, with separatory funnels having features to prevent spillage. Pipets precisely measure fluid amounts, while volumetric flasks are used to create precise solutions by filling to an etched line. All glassware should be cleaned immediately after use.
Laboratory glassware is usually made of borosilicate glass, which is resistant to chemicals except hydrofluoric acid. Common types of glassware include beakers for holding solutions, flasks for heating liquids, volumetric flasks for precisely measuring volumes, and pipettes for dispensing precise amounts of liquids. Pipettes can be graduated or volumetric, and are used to transfer reagents and biological samples in clinical testing.
Microbial Culture Preservation and its MethodsDENNISMMONDAH1
This document discusses various methods for preserving microbial cultures, including short term and long term methods. Short term methods include periodic transfer to fresh media, preservation using saline suspension, drying, and refrigeration. Long term methods discussed are preservation using liquid paraffin/mineral oil, glycerol, lyophilization (freeze drying), and cryopreservation in liquid nitrogen. The aim of preservation is to maintain cultures in viable condition for extended periods without genetic changes.
This document provides an analysis of butter, including its definition, characteristics, apparatus used in analysis, and methods for determining its moisture, fat, curd, and salt content. Butter is defined as a smooth fatty food made from milk or cream that contains at least 80% milk fat. Its characteristics include a firm waxy body and clean cut when sliced. Various apparatus are used like crucibles, filters, flasks, and ovens. Methods described include preparing the butter sample, then determining moisture by heating and weighing. Fat is extracted using petroleum ether, and curd content is calculated. Salt content can be determined using Volhard's or Mohr's titration methods. Microbial contamination sources and controls are also outlined.
The document discusses various staining techniques used to visualize bacteria under a microscope. It describes the principles of simple staining using single dyes like methylene blue or carbol fuchsin. Differential staining techniques like Gram staining and acid-fast staining are also covered, which use multiple dyes to categorize bacteria based on cell wall characteristics. Special stains used to highlight specific bacterial structures such as capsules, flagella or spores are mentioned. Detailed procedures for common staining methods like Gram stain, acid-fast stain and Albert stain are provided. The document aims to explain the use of staining to differentiate bacterial types and visualize their morphology.
This document provides instructions for a laboratory exercise on using the compound microscope, including identifying microscope parts, proper care techniques, calculating magnification, and utilizing different lens powers. It explains that the compound microscope uses multiple lenses to magnify objects beyond what is visible to the naked eye. Proper use of immersion oil and understanding numerical aperture allow for gathering more light and achieving better image resolution under the microscope.
This laboratory report summarizes three experiments conducted by a group of students:
1. Media preparation demonstration - The group prepared two types of nutrient agar media.
2. Isolation of soil bacteria - The group determined the viable titer of soil bacteria by serial dilution and plating, then isolated pure cultures.
3. Staining techniques - The group demonstrated endospore staining of Bacillus and gram staining of an unknown bacterium to identify cell structure.
Lab Report: Isolation of Pure Culture, Gram-staining, and Microscopic Observa...Annisa Hayatunnufus
A Lab Report under the subject of Microbiology. Done as a lab session in Josai University, Japan during a twinning program on 2014.
Created by: Annisa Hayatunnufus
Bachelor of Pharmacy
Management & Science University
The document provides instructions for students on the proper use and care of a microscope during laboratory experiments. It describes the main components of the microscope, including the body, base, stage, illumination system, magnifying lenses, and adjusting system. It provides step-by-step instructions on how to use the different objective lenses, adjust the light and focus to view unstained and stained samples. Students are advised to clean lenses after use and store the microscope properly.
This document provides information about lab exercises on gram staining, endospore staining, and capsule staining. It includes the procedures for each stain and discusses what structures each stain targets (e.g. gram stain targets the peptidoglycan cell wall). It also provides background on Bacillus anthracis and how it can cause disease. Key points covered are that the gram stain differentiates bacteria types, endospore stain uses malachite green to stain spores, and the capsule stain demonstrates capsules using Congo red and Maneval's solution.
The document provides an overview of microscopy, including definitions, the historical background, key variables, and types of microscopes. It describes the compound microscope's structure and functions, including the ocular lens, body tube, nose piece, objectives, stage, diaphragm, illumination, and controls. The document also discusses magnification, resolution, numerical aperture, aberrations, Kohler illumination, and provides examples of different microscope types.
This document provides information about gram staining, including:
1) Gram staining is a method developed by Hans Christian Gram that classifies bacteria as either Gram-positive or Gram-negative based on their ability to retain crystal violet dye. Gram-positive bacteria retain the purple crystal violet dye after alcohol washing, while Gram-negative bacteria appear red.
2) The document outlines the principle of gram staining, noting that basic dyes like crystal violet bind to the slightly negatively charged bacterial cells, while acidic dyes stain the background.
3) The requirements and step-by-step method for performing a gram stain are provided, including heat fixing a bacterial sample on a slide, applying crystal violet and
1. Water demand comes from domestic, industrial, and public uses. Domestic demand includes residential use for drinking, bathing, cleaning, and outdoor uses like gardening. Industrial demand is for processes, fabrication, washing, and cooling. Public demand incorporates institutional use by places like hospitals and schools, as well as commercial needs in offices, restaurants, and other businesses.
2. Population growth and changes in lifestyle are increasing demand, especially in urban areas. Climate change may also impact water security by increasing uncertainty in supply.
3. Specific domestic uses are interior like taps and toilets, and exterior like lawns. Institutional use serves facilities. Commercial demand provides for employee and customer needs as well as special processes
This lab report summarizes an experiment comparing fresh and 2-day old yogurt samples through gram staining of bacteria. On the first day, all bacteria from the refrigerated yogurt sample stained gram-positive. On the second day, the unrefrigerated yogurt developed both gram-positive and gram-negative bacteria, as observed through visual analysis of staining, though human observation has limitations. The experiment had to be repeated for some groups that did not initially observe gram-negative bacteria, likely due to sampling from lower parts of the yogurt container rather than the top surface exposed to air.
This document summarizes 6 workshops attended by the author:
1) A microscopy workshop where techniques like phase contrast were learned to view organisms.
2) A workshop on micropipetting and aseptic techniques like streak plating that are crucial in biology labs.
3) A workshop from UNC on DNA extraction, PCR, gel electrophoresis, and SDS-PAGE that demonstrated techniques from DNA to protein.
4) A workshop where lysozyme protein was purified from egg whites using chromatography and SDS-PAGE.
5) A neurobiology workshop from MSU that explored the sensory and motor systems and how the brain works.
6) A workshop on using protein structure to
This document provides instructions for performing a Gram stain procedure. The Gram stain allows bacteria to be classified as either Gram-positive or Gram-negative based on differences in their cell wall structure and how they interact with stain reagents. The procedure involves staining a bacterial smear with crystal violet, applying Gram's iodine as a mordant, decolorizing with ethanol or acetone, and counterstaining with safranin. Gram-positive bacteria appear purple or violet while Gram-negative bacteria appear pink or red. The document provides step-by-step instructions for performing the Gram stain and interpreting results under the microscope.
This document provides an introduction and guidelines for a medical microbiology laboratory course. It outlines the learning objectives and expectations for each lab exercise. Strict safety procedures are reviewed, including proper disposal of materials and hand washing. Guidelines are provided for laboratory conduct, including maintaining an organized work area and following instructions. The document also reviews basic microbiology topics like binomial nomenclature and metric units commonly used in measurements of microorganisms.
Pure cultures are important in microbiology because they allow for the accurate study and identification of microorganisms. There are three main techniques involved in obtaining a pure culture: sterilization of materials to prevent contamination, aseptic transfer of microbes to growth media, and isolating single cells or their progeny. Some common isolation methods are streak plating, spread plating, and serial dilution plating. Once a pure culture is obtained, its purity can be demonstrated by the uniform appearance of colonies and identical growth characteristics of isolated colonies. Pure cultures must then be maintained through refrigeration, paraffin coating, cryopreservation, or lyophilization to preserve them for long-term storage and future use.
This document provides information about interpreting Gram stains and describes the morphology of various bacteria under the Gram stain. It begins by explaining the Gram stain procedure and how to judge the quality of a Gram stain. It then describes the appearance of many Gram-positive and Gram-negative bacteria including cocci, rods, fungi and some human cells. Key details about cell arrangement, shape, size and other distinguishing features are provided for common genera like Staphylococcus, Streptococcus, Bacillus, Pseudomonas and Neisseria.
Subit Barua New Haven, Connecticut
Department of Microbiology and Immunology
University of Maryland School of Medicine Trinad Chakraborty
Baltimore, Maryland Department of Microbiology
University of Kiel
Sukhadeo Barbuddhe Kiel, Germany
Department of Microbiology
University of Kiel Rita R. Colwell
Kiel, Germany University of Maryland Biotechnology Institute
College Park, Maryland
Michael Bott
Institute for Biotechnology Jongsik Chun
Research Centre Juelich Department of Microbiology and Molecular Genetics
Juelich, Germany University of Maryland
College Park, Maryland
Paramita Basu
Department of Microbiology and Immun
El documento describe al águila real, una especie en peligro de extinción. Mide hasta 1 metro de largo y 2.2 metros de envergadura. Sólo quedan 70 parejas debido a la pérdida de hábitat por actividades humanas. Es un depredador que caza desde el aire para alimentarse de mamíferos y aves pequeñas. Se reproduce de forma solitaria, poniendo 1-4 huevos por temporada.
The document is a contents page for a school magazine that lists the sections and page numbers. It includes a main image of a girl holding her exam results as well as additional images highlighting events and trips covered in the magazine.
The document discusses various staining techniques used in microbiology, including Gram staining, acid-fast staining, and simple staining techniques. Gram staining differentiates bacteria into gram-positive and gram-negative groups based on differences in their cell wall structure and how they retain or release crystal violet dye. Acid-fast staining uses a carbolfuchsin primary stain to identify acid-fast bacteria that resist decolorization by acid-alcohol, such as Mycobacterium tuberculosis. Simple stains like Loeffler's methylene blue and diluted carbol fuchsin are also discussed, which provide contrast but do not differentiate bacterial types.
The document discusses various staining techniques used in microbiology, including Gram staining, acid-fast staining, and simple staining techniques. Gram staining differentiates bacteria into gram-positive and gram-negative groups based on differences in their cell wall structure and how they retain or release crystal violet dye. Acid-fast staining uses a carbolfuchsin primary stain to identify acid-fast bacteria that resist decolorization by acid-alcohol, such as Mycobacterium tuberculosis. Simple stains like Loeffler's methylene blue and diluted carbol fuchsin are also discussed, which provide contrast but do not differentiate bacterial types.
This document describes various staining techniques used to visualize bacteria under a microscope. It discusses how stains work by imparting color to bacteria and cellular structures. Specific stains mentioned include Gram stain, acid-fast stain, simple stains like Loeffler's methylene blue. The Gram stain technique colors bacteria either purple (Gram positive) or red (Gram negative) depending on their cell wall structure. The acid-fast stain uses carbolic fuchsin and retains its color after acid alcohol treatment to identify acid-fast bacteria like Mycobacterium tuberculosis. Proper staining allows clear visualization of bacterial morphology and structures.
Staining techniques are used in microbiology to identify bacteria under a microscope. There are several types of staining including simple staining with one dye, Gram staining which differentiates bacteria as Gram-positive or Gram-negative based on cell wall structure, and acid-fast staining used to identify Mycobacterium species. Biochemical tests such as IMViC (Indole, Methyl Red, Voges-Proskauer, Citrate) are also used to identify bacteria based on their metabolic reactions and products.
This document provides information about identification of bacteria through staining and biochemical tests. It discusses various staining techniques like simple staining, Gram staining, negative staining and acid fast staining. It explains the principles, procedures and results of these staining methods. It also describes the IMViC tests, which are used to identify Gram negative bacteria based on their ability to produce indole, change the color of methyl red, produce acetoin in Voges-Proskauer test and utilize citrate as a carbon source. Understanding these staining techniques and biochemical tests is important for identification of microbes.
1. Gram staining is a differential staining technique developed by Hans Christian Gram in 1884 that is used to classify bacteria into two groups: Gram-positive and Gram-negative.
2. The key steps of Gram staining involve staining with crystal violet dye, treating with iodine, decolorizing with alcohol or acetone, and counterstaining with safranin.
3. Gram-positive bacteria retain the crystal violet dye after decolorization due to their thick peptidoglycan cell wall, while Gram-negative bacteria lose the dye due to their thinner cell wall. This allows bacteria to be classified based on their staining.
Hans Christian Gram, a Danish physician and bacteriologist, developed the Gram staining technique in 1884 to classify bacteria. The technique involves staining a smear of bacteria with crystal violet dye, washing with iodine to form a crystal violet-iodine complex within the cell wall, then decolorizing with alcohol or acetone. Gram-positive bacteria retain the crystal violet due to their thick peptidoglycan layer, appearing dark purple under the microscope. Gram-negative bacteria lose the crystal violet during decolorization due to their thin peptidoglycan layer and outer membrane, appearing red with the counterstain. The Gram stain technique is a simple yet effective way to rapidly classify bacteria and guide
This document provides information about dyes, stains, and staining methods used in microscopy. It begins with definitions of stains and dyes, explaining that stains are used to facilitate examination under a microscope while dyes are compounds that impart color. It then classifies biological stains as acidic, basic, neutral, metachromatic, or fluorescent based on their chemical properties. Various staining techniques are described including simple staining, negative staining, Gram staining, and acid-fast staining. Specific staining methods and the mechanisms of how they work are explained for simple staining, Gram staining, and acid-fast staining.
This document provides information about dyes, stains, and staining methods used in microscopy. It begins with definitions of stains and dyes, explaining that stains are used to facilitate examination under a microscope while dyes are compounds that impart color. It then classifies biological stains as acidic, basic, neutral, metachromatic, or fluorescent based on their chemical properties. Various staining techniques are described including simple staining, negative staining, Gram staining, and acid-fast staining. Specific staining methods and the mechanisms of how they work are explained for simple staining, Gram staining, and acid-fast staining.
This document discusses various staining techniques used to visualize bacteria under a microscope. It covers simple staining techniques like Gram staining and acid-fast staining, as well as methods to identify specific structures like volutin granules and bacterial spores. Gram staining uses dyes to differentiate between Gram-positive and Gram-negative bacteria based on their cell wall composition. Acid-fast staining targets bacteria with thick lipid cell walls like Mycobacterium tuberculosis. Specialized techniques employ unique dyes and fixation steps to highlight intracellular inclusions and endospores. Proper staining is crucial for bacterial identification and clinical diagnosis.
this presentation involves a comprehensive outlines regarding the most common different methods used in diagnostic microbiology to stain bacteria and their structures
INTRODUCTION TO MICRO LAB, STAINING TECHNIQUES & MORPHOLOGY OF BACTERIADrBhavikapatel
This PPT is helpful to understand first practical to 2nd year MBBS student.
I have added 2 video in this PPT to understand staining techniques properly.
Reference: 1 Gram stain video: Dr.G Bhanu prakash animated medical videos
2. Zn stain video: sridhar Rao
This document provides an introduction to lab techniques for staining and visualizing microorganisms under a microscope. It discusses the purpose of staining to improve contrast and differentiate morphological characteristics. Various staining techniques are described including simple staining using one dye, differential staining using two contrasting dyes, and special staining methods for visualizing structures like capsules and endospores. Gram staining and acid-fast staining techniques are explained in detail including the chemical processes involved and appearance of stained microorganisms.
This document discusses various staining techniques used to visualize microorganisms under the microscope. It describes two main types of staining: positive staining, which colors the microorganisms, and negative staining, which colors the background. Specific staining methods covered include simple staining using single dyes, differential staining techniques like Gram staining and acid-fast staining, and special stains for structures such as endospores, capsules, flagella, and nuclei. Detailed procedures are provided for common staining methods along with labeled microscope images showing the results.
This document discusses various methods for identifying unknown bacterial cultures, including phenotypic, immunological, and genetic techniques. It focuses on morphological identification methods such as staining techniques like simple staining, negative staining, Gram staining, and acid-fast staining. These staining methods allow observation of bacterial size, shape, arrangement and properties to determine the taxon. Identification is important for medical, industrial, and research applications.
This document discusses various bacterial staining techniques including simple stains that use a single dye, differential stains that use a primary and counterstain separated by a decolorizing agent, and special stains for specific bacteria. The most common differential stain is the Gram stain, which distinguishes between Gram-positive and Gram-negative bacteria based on their cell wall structure. Another important differential stain is the Ziehl-Neelsen stain used to identify acid-fast bacteria like Mycobacterium tuberculosis through their mycolic acid cell walls. Special stains are also described that target specific bacteria like the Fontana stain for spirochetes.
- The Gram staining technique was developed in 1884 by Hans Christian Gram as a way to classify bacteria.
- Gram staining involves staining a bacterial smear with crystal violet dye followed by iodine to form a crystal violet-iodine complex. Bacteria are then decolorized with alcohol or acetone and counterstained with safranin.
- Based on whether they retain the crystal violet dye after decolorization, bacteria are classified as either Gram-positive or Gram-negative. Gram-positive bacteria retain the crystal violet due to their thick peptidoglycan cell wall, appearing purple under the microscope, while Gram-negative bacteria do not retain the dye due to their thinner cell wall
This document discusses various methods used to identify unknown bacterial cultures, which is a major responsibility of microbiologists. It outlines staining techniques like Gram staining, acid-fast staining, endospore staining, and capsule staining. These techniques examine morphological characteristics of bacteria like shape, arrangement, presence of spores or capsules. The document also mentions biochemical tests that detect bacterial enzymatic activity or ability to ferment carbohydrates and produce acids/gases. Identifying pathogenic bacteria is important for medical diagnostics and food/brewing industries to prevent contamination.
The document discusses bacterial staining techniques, specifically simple staining and Gram's staining. It begins by explaining how staining enhances contrast under the microscope since bacteria are otherwise invisible. It then describes the basic components and process of simple staining, as well as the principles and steps of Gram's staining technique. Gram's staining allows differentiation of bacteria into Gram-positive or Gram-negative categories based on differences in cell wall structure and composition. This differential staining technique is one of the most common and important in microbiology.
This document discusses different staining techniques used to visualize bacteria under a microscope. It describes simple staining using single dyes like methylene blue, and differential staining techniques like Gram staining and acid-fast staining. Gram staining differentiates bacteria into Gram-positive and Gram-negative groups based on their ability to retain or lose crystal violet dye. Acid-fast staining is used to identify acid-fast bacteria like Mycobacterium that appear bright red after staining. These staining methods allow clear visualization of bacterial morphology and structure.
Similar to bacterial different staining technique (20)
Muktapishti is a traditional Ayurvedic preparation made from Shoditha Mukta (Purified Pearl), is believed to help regulate thyroid function and reduce symptoms of hyperthyroidism due to its cooling and balancing properties. Clinical evidence on its efficacy remains limited, necessitating further research to validate its therapeutic benefits.
Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
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Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
1. LAB 3
Bacterial Staining Techniques II
I. Differential Stains: Gram Stain and Acid-fast Stain
II. Morphological Unknown
I. DIFFERENTIAL STAINS
A. Gram Stain
B. Acid-fast Stain
A. Gram Stain
The previous lab introduced simple staining techniques that enable microbiologists to observe the
morphological characteristics of bacteria. Although simple stains are useful, they do not reveal details
about the bacteria other than morphology and arrangement. The Gram stain is a differential stain
commonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wall
structure. Most bacteria can be divided into two groups based on the composition of their cell wall:
1) Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane.
Characteristic polymers called teichoic and lipoteichoic acids stick out above the peptidoglycan
and it is because of their negative charge that the cell wall is overall negative. These acids are
also very important in the body’s ability to recognize foreign bacteria. Gram-positive cell walls
stain blue/purple with the Gram stain.
2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outer
membrane beyond the plasma membrane. The space between the plasma membrane and the outer
membrane is called the periplasmic space. The outer leaflet of the outer membrane is composed
largely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important in
triggering the body’s immune response and contributing to the overall negative charge of the cell.
Spanning the outer membrane are porin proteins that enable the passage of small molecules.
Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells will
stain pink with the Gram stain.
This is The Most Important staining technique in Bacteriology.
Cell wall structure of Gram+ and Gram-
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2. GRAM STAIN
Cell Color
Procedure Reagent Gram Positive Gram Negative
Fixed cells on slide COLORLESS COLORLESS
Primary stain Crystal Violet PURPLE PURPLE
Mordant Iodine PURPLE PURPLE
Decolorizer Alcohol PURPLE COLORLESS
Counterstain Safranin PURPLE RED
An easy way to remember the steps of the Gram stain is...
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3. PROCEDURE: (EACH STUDENT)
1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a mixed smear of
Escherichia coli (G- rod) and Staphylococcus epidermidis (G+ coccus).
2. Air dry and Heat fix.
3. Cover the smear with Crystal Violet (primary stain) for 1 min.
4. Gently wash off the slide with water.
5. Add Gram’s Iodine (mordant) for 1 min.
6. Wash with water.
7. Decolorize with 95% ethanol. This is the "tricky" step. Stop decolorizing with alcohol as soon as the
purple color has stopped leaching off the slide (time will vary depending on thickness of smear).
Immediately wash with water. Be sure to dispose of all ethanol waste in the appropriately labeled waste
container.
8. Cover the smear with Safranin for 30 seconds.
9. Wash both the top & the bottom of the slide with water.
10. Blot the slide with bibulous paper.
11. Using the 10X objective lens, focus first on the line and then on the smear. Follow the focusing
procedure in Lab #1. Use the focusing procedure in Lab #2 to view the smear using the 100X (oil
immersion lens).
Focus line
E. coli
S. epidermidis
Note: Escherichia coli is a tiny pink (Gram-) rod. Staphylococcus epidermidis is a purple (Gram+) sphere
or coccus.
Draw a picture of a typical microscopic field and identify both Escherichia coli and Staphylococcus
epidermidis. Record this in the results section for this lab. Colored pencils are available throughout the room
on the chalkboard trays.
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4. B. Acid-fast Stain
Mycobacterium and many Nocardia species are called acid-fast because during an acid-fast staining
procedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solvent
acid-alcohol (95% ethanol with 3% HCl). Nearly all other genera of bacteria are nonacid-fast. The acid-
fast genera have the waxy hydroxy-lipid called mycolic acid in their cell walls. It is assumed that
mycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differential
stain.
Ziehl Neelsen Acid-fast stain
ACID-FAST STAIN
Cell Color
Procedure Reagent Acid-fast Bacteria Nonacid-fast Bacteria
Primary dye Carbolfuchsin RED RED
Decolorizer Acid-alcohol RED COLORLESS
Counterstain Methylene blue RED BLUE
PROCEDURE - (EACH STUDENT)
1. Add one loopful of sterile water to a microscope slide.
2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your loop. Then transfer a
small amount of Staphylococcus epidermidis to the same drop of water.
You will now have a mixture of M. smegmatis and S. epidermidis.
3. Air dry and heat fix well.
4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on top of the dye. Be sure the
paper towel is saturated with the dye. Carbolfuchsin is a potential carcinogen. Please wear gloves when
working with this dye.
5. Place the slide on the rack over dry heat for 2 minutes.
6. Cool and rinse with water.
7. Decolorize by placing a drop of acid alcohol on the slide and allowing it to sit for 15 seconds.
8. Wash the top and bottom of slide with water and clean the slide bottom well.
9. Counterstain with Methylene Blue for 30 seconds to 1 minute.
10. Wash and blot the slide with bibulous paper.
11. Focus 10X - then use oil immersion.
M. smegmatis (heavy)
Focus line
S . epidermidis
Draw a typical microscopic field and record in Results Lab 3.
Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The Staphylococcus
epidermidis is decolorized and the counterstain colors them blue. See table for acid-fast stain.
24
5. II. Morphological Unknown: continue your investigation of your morphological unknown.
1. Collect your unknown from the side bench. Verify that the # matches that used for your direct stain.
2. Perform a Gram stain and an Acid-fast stain as described in the above sections.
3. Record your observations in the Results section of Lab #4.
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7. LAB 3 RESULTS
I. DIFFERENTIAL STAINS
A. Gram Stain Draw and label examples of Escherichia coli and Staphylococcus epidermidis.
B. Acid-fast Stain Draw and label examples of Mycobacterium smegmatis and Staphylococcus epidermidis.
Mycobacterium & Staphylococcus
smegmatis epidermidis
QUESTIONS:
1. What is the difference between a simple and a differential stain?__________________________
_______________________________________________________________________________
_______________________________________________________________________________
2. Describe the function of each of the following in the Gram stain
Mordant: __________________________________________________________________
Primary stain:_______________________________________________________________
Decolorizer: ________________________________________________________________
Counterstain: _______________________________________________________________
3. Which step in the Gram stain is most likely to cause poor results if done incorrectly? _________
_______________________________________________________________________________
4. Why must fresh bacterial cultures be used in a Gram stain? ______________________________
_______________________________________________________________________________
27
8. 5. Briefly describe the mechanism of Gram staining. _____________________________________
_______________________________________________________________________________
_______________________________________________________________________________
6. What is the primary stain used in the acid-fast staining procedure? ________________________
_______________________________________________________________________________
7. What is the purpose of the heat/steam during the acid-fast staining procedure? _______________
_______________________________________________________________________________
_______________________________________________________________________________
8. In a clinical microbiology laboratory, the acid-fast stain would be used for diagnosis of what diseases?
_______________________________________________________________________________
9. What makes a microorganism nonacid-fast? __________________________________________
_______________________________________________________________________________
Organisms introduced in this lab:
Mycobacterium smegmatis
Mycobacterium tuberculosis
Mycobacterium leprae
Staphylococcus epidermidis
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9. GENERAL MICROBIOLOGY 2210: GRAM STAIN REPORT (15 PTS)
ASSIGNMENT
Each student will have the opportunity to submit one example of a Gram stain with a brief written report
describing the staining procedure and the stain itself. A good report will likely necessitate a minimum of 2
pages double-spaced and is limited to a 3-page maximum.
OVERALL FORMAT
Please include the following in your report:
A. Title Page with the following information: (1 point) __________
--Title: A title should be brief, creative, but descriptive of the work that was done.
--Your Name
--Course and Lab Section
--Date
B. Introduction: (4 points) __________
An introduction should include pertinent background information. In this case, the history of the Gram
stain and its significance in microbiology are relevant (1 point). A discussion of the mechanism of Gram
staining and how it differentiates bacteria on the basis of their cell wall structure should also be included
(1 point). The purpose and objectives of the experiment should be stated and a hypothesis should be
made (2 points). Remember that a hypothesis need not be correct, but it must be tested by the procedure.
Good hypotheses are also grounded in background knowledge and clearly state the predicted results.
Hypotheses can be of several types. 1 We will discuss two types here:
1. Manipulated hypotheses
This type of hypothesis is used when one variable (the independent variable) is manipulated by the
experimenter and the effects on a second variable (the dependent variable) are observed. This type of
hypothesis is generally written as an if, then statement (e.g. If the smear of the unknown bacterium is
decolorized as according to the above procedure (neither over- nor under-decolorized), then all the
cells will stain a single color.) In this example, decolorization is the independent variable (the
variable controlled by the experimenter) and the color of the cells is the dependent variable (the
variable affected by the changes in the independent variable).
2. Observational hypotheses
States something about the nature of an organism (e.g. The unknown bacterium will stain Gram-
positive.)
C. Materials and Methods: (4 points) __________
The Materials and Methods section should include a description of the procedure/steps in the Gram stain,
including the preparation of a bacterial smear and heat fixation (3 points). This section must be written
in complete sentences and in paragraph form (1 point). Illustrations may assist in clarifying points.
D. Results: (1 point) __________
In the results section, describe your Gram stain/s. Include at least one illustration of your own
creation.
E. Discussion: (3 points) __________
What features of your Gram stain exemplify the Gram stain theory (1 point)? If there are features of the
Gram stain that are not as expected, what possible sources or error may be responsible (e.g. poor quality
of the smear preparation)? (1 point) Discuss whether the hypothesis made in the introduction was tested
and if so was it supported? (1 point)
1
For a discussion of hypotheses formation please visit
http://www.utas.edu.au/sciencelinks/exdesign/HF2C.HTM
29
10. OVERALL QUALITY
Students have the opportunity to earn points for reports that are well written, organized and edited.
(2 points) __________
DUE DATE: TAs will inform you of the due date.
*Please tear this grading key out of your lab book and attach it to your lab report at the time of
submission. Please also write your name and lab section on the frosted portion of your Gram stain
slide and hand it in with your written report.
TA comments: __________________________________________________________________________
_______________________________________________________________________________________
_______________________________________________________________________________________
TA’s suggestions for improving Gram stain: ___________________________________________________
_______________________________________________________________________________________
_______________________________________________________________________________________
Points earned out of 15 possible: ____________
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