Microorganism Staining Techniques

• Live bacteria does not reveal structural detail under a light microscope due to lack of contrast
• Standard methods for staining microorganisms include drying and fixing smears, killing the microorganisms in the process
• Bacteria have a preference for basic dyes due to acidic nature of the protoplasm

Most Popular Staining Methods

• Simple Staining
• Negative Staining
• Differential Staining: Gram Staining
• Differential Staining: Acid-fast Staining
• Structural Staining: Spore Stain

Simple Staining

• Are developed using dyes like basic fuchsin or methylene blue
• Offers color contrast while giving all microorganisms the same color

Negative Staining

• Capsule is a type of protective outer shell seen on some bacteria and yeasts
• Knowing whether cells have capsules is an important diagnostic technique, since capsule existence is directly tied to a microbe's pathogenicity (disease capacity)
• Coloring the surrounding cells using a negative staining approach is utilized to stain capsules since most common dyes are not absorbed by capsules
• Capsules round the cell like haloes and are unaffected by the dye, which only dyes the background
• Negative staining does not require heat-fixing the sample
• Dyes such as Indian ink or nigrosin are added to the sample to create a background that is consistently colored and contrasted with the unstained bacteria
• Allows for observation of very thin microorganisms, such as spirochetes, which cannot be seen with conventional staining techniques
• India ink was used to color the area surrounding Cryptococcus neoformans cells in the sample of a negative stain in Figure 6.1
• Polysaccharide capsules are seen as halos surrounding the cells

Differential Staining: Gram Staining

• The two most commonly used differential stains are Gram stain and Acid-Fast stain
• These stains give distinct bacteria or bacterial structures different colors
• Histologist Christian Gram introduced the gram stain to stain bacteria in tissues
• Gram-positive cells have an acidic protoplasm
• Gram-positive cells possess a considerable layer of peptidoglycan
• Teichoic acids are entangled with peptidoglycans and serve as the primary determinants of surface antigens
• Gram-negative cells are more intricate and lipid-rich
• Phospholipids, proteins, and lipopolysaccharides make up the Gram-negative cell's membrane bilayer
• Endotoxin is another name for lipopolysaccharides (LPS)
• The peptidoglycan layer of Gram-negative cells is very thin, made of only one or two molecules
• Gram-negative bacteria have no Teichoic acids in their cell walls
• Solutes are transferred across lipopolysaccharide channels with porins in the outer membrane
• Bacterial morphology studies typically involve the use of lipoprotein, which connects the outer membrane and peptidoglycan layer of the bacteria
• Permeability of the bacterial cell wall and cytoplasmic membrane to the dye-iodine complex in Gram-negative, but not Gram-positive, cells may be related to the Gram reaction
• The complex is allowed to exit during decolorization
• Gram staining is a crucial step in identifying bacteria and is frequently the only technique needed to examine their morphology

Bacterial Cell Shape and Arrangement

• Cocci are spherical or oval cells and comes from the Greek word for berry
• Bacilli means "rod cells" in Latin and are rod-shaped cells
• Vibrios are curved rods with a comma form that get their name from their vibratory movement
• Spirilla are round and stiff shaped
• Spirochetes are flexuous spiral structures derived from speira (coil) and chaite (hair)
• Actinomycetes are branching filamentous bacteria that resemble the sun's radiating beams when they are found in tissue lesions and is derived from the words actis, meaning ray, and mykes, meaning fungus
• Mycoplasmas are bacteria that lack a stable shape due to a deficiency in their cell walls, which appear as interlacing filaments with circular or oval bodies
• Bacteria occasionally display distinctive cellular grouping or organization
• Cocci can be grouped in pairs (diplococci), chains (streptococci), groups of four (tetrads), eight (sarcina), or grape-like clusters (staphylococci), depending on the plane of cellular division

Differential Staining: Acid-fast staining

• Ehrlich discovered the acid-fast stain when he observed that tubercle bacilli can withstand acidic decolorization after being stained with aniline dyes
• The technique, as updated by Ziehl and Neelsen, is currently widely used
• The term "acid fast" comes from the finding that certain stained cells still maintain the primary stain after being treated with hydrochloric acid and alcohol decolorizer (carbolfuchsin)
• After the counterstaining process is finished, cells that release the primary stain (carbolfuchsin) with decolorizing will be visible
• Bacteria that are described as acid-fast will appear red when samples are looked at under bright-field microscopy
• Field debris and non-acid-fast cells will appear blue

Structural Staining: Spore Stain

• Endospore staining requires two stains to distinguish endospores from the rest of the cell
• The primary stain is malachite green, which is pushed into the endospore using heat in the Schaeffer-Fulton method
• The Schaeffer-Fulton method is the most widely used endospore-staining technique
• The cell loses its color while the endospore retains the green stain after washing with water
• Safranin is used to apply a pink counterstain to the cell
• The resulting image displays the shape and position of endospores if they are present
• The pink vegetative cells will either contain the green endospores or they will seem entirely isolated from the pink cells
• Only the pink vegetative cells will be visible if there are no endospores
• Examples of positive endospore staining include Clostridium perfringens, C. botulinum, C. tetani, Bacillus anthracis, Bacillus cereus, Desulfotomaculum spp., Sporolactobacillus spp., and Sporosarcina spp
• The position and shape of spores can be central and spherical (not bulging), subterminal and elliptical (bulging), or terminal
• Keywords for cell staining; Counterstain, Decolorize, Heat-fix, Differential stain, Mordant, Negative stain, Parfocal, Primary stain, Simple stain, Smear, Vegetative cell, Endospore, Conidia, Sporogenesis, Germination

Objectives of cell staining

• Prepare smears of bacteria

• Perform staining technique such as simple staining and negative staining skillfully

• Distinguish changes in morphological characteristics based on reactions with differential stains

• Describe the morphological features of bacteria

• Utilize the oil-immersion objective of the compound light microscope with the appropriate skills

• The materials and reagents required are: Stock cultures (bacteria), Crystal violet (simple stains), Nigrosin, India ink, or Congo red (negative stains), Crystal violet, methylene blue, or safranin (basic stains), Crystal violet, Gram-iodine, 95% ethanol, safranin (Gram stain), Lactophenol, Wash bottle, Clear adhesive tape, Compound light microscope, Lint-free tissue paper, Bunsen burner or alcohol lamp, Clothespin, Carbolfuchsin, acid-alcohol, methylene blue (acid-fast stain), Glass slides and cover slips, Beaker with water, Immersion oil, Inoculating loop and needle, Staining tray, Gum labels and permanent marking pens, Clean Type, Clear nail polish

• Staining procedure; Label slide at one end (i.e. on the frosted portion), which is unlikely to contact the stain with Organism used, Staining method used, Name of student

• When staining use clean, grease free, dry slides as well as to save space, abbreviations for organism and staining method may be used

• Use a sterile and cooled loop to transfer loopfuls onto the slide and to spread out the smear on the blank portion of the slide with the smear air-dried

• Heat-fix by passing the slide film sideways three to five times over the flame using a slide holder or clothespin

• Put a small drop of water on the slide using a wash bottle for the agar cultures

• Mix a small amount of growth with the drop of water on the slide with a sterile and cooled loop

• Spread it evenly with your loop, air-dry, and heat-fix

• Make sure to flame-sterilize the loop before and after using it, that too much broth culture on the slide would delay the air-drying process, that heat-fixing serves to make the cells adhere to the slide, that you prepare smears that are neither too thick nor too thin since thick smears will be unevenly stained and will be tough to see individual cells

• Prepare a smear of 24-48- hour broth or agar culture of Pseudomonas aeruginosa and Staphylococcus aureus

• Cover the smear with crystal violet for 1 minute using a dropper

• Tilt the slide and pour off the stain into an empty beaker after 1 minute

• Rinse off the remaining stain using a wash bottle with the slide tilted

• Carefully blot-dry the smear with tissue paper

• Examine cells under high-power objectives (HPO) and oil-immersion objectives (O10)

• Place a drop of any negative stain near the edge of a clean glass slide, aseptically obtain a loopful of broth cultures, transfer the loopful of culture to the drop of stain on the slide and mix the culture into the drop

• Always flame your loop before setting it down. with a second slide get a 45° angle with the first slide

• The bacteria-stain suspension spreads along the back edge of the slide

• Spread the suspension until the surface area of the slide is covered

• Allow air-drying, the stained material should form a thin film then examine the slides under HPO and OIO by remembering do not wipe-dry to avoid removing the cells from the slide as well as do not wipe the oil off the slide before you submit it as there is no need to put a cover slip on

• Prepare bacterial smears of 18- to 24-hour broth & agar cultures of Bacillus sp. and Enterobacter aerogenes

• Cover the smear with crystal violet for 1 minute using a dropper, drain and rinse with water, tap the excess water from the slide onto a piece of tissue paper

• Using another dropper, cover the smear with Gram-iodine for 1 minute, drain and rinse with water & tap the excess water

• Use another dropper to decolorize with 95% ethanol until no more violet stain runs off and tilt the slide as you do this as well as rinse afterward

• Use another dropper to cover the smear with safranin and after 1 minute, drain and rinse

• Blot-dry and examine under OIO noting that Gram-positive cells are blue to purple, while Gram-negative cells are pink to red Remember:

• Do not overstain and that timing is very critical in Gram staining noting to Decolorize for 10-15 seconds only

• Prepare a smear of 5-7-day cultures of Mycobacterium sp. and P. aeruginosa using the acid-fast Staining (Ziehl-Neelsen Acid-fast Staining) methodology

• Apply carbolfuchsin to saturate the paper, and heat for 5 minutes. NOTE: The carbolfuchsin heated using either a hot plate or a Bunsen burner flame, in either case, gently steam only; do not boil.

• As the paper dries out adding more stain to keep the paper moist

• After 5 minutes removing the paper, cool, and then rinse with water for 30 seconds

• Decolorize with acid-alcohol until pink (10-30 seconds) then rinse with water for 5 seconds using the counterstain with methylene blue for about 2 minutes

• Rinse with water for 30 seconds then blot dry with tissue paper and examine under O10

• Prepare a smear of Bacillus sp. and E. coli (4-5-day cultures), again, from agar culture using the Spore Staining (Schaeffer-Fulton Endospore Staining)

• Cover the smear with malachite green and let it steam over the flame for 5 minutes reminding NOT to allow the stain to boil or dry up as well as to replenish the stain as needed, and to use your clothespin to hold the slide

• After the 5-minute steaming. allow the slide to cool down first then drain off excess stain in an empty beaker and rinse with water until no more green stain runs from the slide

• Tap the excess water on a piece of tissue paper covering it with the smear with safranin counterstain for 1 minute & then drain and rinse as before blotting dry and examining under O10 using NOTE where spores or endospores appear as spherical green structures, and the vegetative cells in rod-shaped red-colored cells remembering that you be careful not to stain your fingers with malachite green & if cultures are very old (48 hours old), one the exospores are visible and the vegetative cells may no longer be visible as they have been converted into spores

• List the objectives of determining the Morphological & Biochemical Characterization of Yeasts:

  • Distinguish changes in morphological characteristics based on reactions differential staining for the cell wall
  • Describe the morphological and biochemical features of yeasts
  • Describe yeast metabolism using different substrates as well as analyze metabolic processes in yeasts using different substrates

• Yeasts are microscopic fungi in the form of cells that reproduce by cellular budding

• Molds, also a fungi, appear in filaments that grow by extension and reproduce through their spores

• The morphological characterization of yeasts involves the determination of the cells' form, shape, and size, which are special structures unique to a particular species

• Yeasts' morphological and cultural characteristics are vital in identifying and differentiating other organisms, especially in disease diagnosis

• Yeasts are chemoorganotrophs that obtain carbon from hexose sugars, such as glucose and fructose, or from disaccharides such as sucrose and maltose

• Some species of yeast can metabolize pentose sugars, alcohols, and even organic acids

• Some species may either require oxygen (obligate aerobes) that they use for cellular respiration, while others may not require oxygen (facultative anaerobes) but have an aerobic type of energy production, which is used in biotechnology

• Rapid identification kits can also be used with specific sets of commercially available biochemical tests, such as API® 20C AUX, to identify species of yeasts based on their carbohydrate assimilation capacity using different substrates of carbohydrates

• API is used to classify species of yeasts based on miniaturized biochemical reactions of microbiological cultures based on the manufacturer's protocol

• Specific genotypic and proteomic technologies have also been developed to identify microorganisms down to the species level Keywords:

  • Counterstain
  • Decolorize
  • Heat-fix
  • Cell wall stain
  • Carbohydrate assimilation

• Objectives: prepare smears of yeast, and perform staining techniques for yeasts

  • Materials and Reagents include: Stock cultures (bacteria), Crystal violet (simple stains), Methylene blue, Gram-iodine, Wash bottle, Clear adhesive tape, Compound light microscope, Lint-free tissue paper, Beaker with water, Immersion oil, Inoculating loop and needle, Test tubes, Resazurin, Yeast nitrogen base (YNB) media, Peptone, Glycerol, 2-keto-D-gluconate, D-xylose, Xylitol, Inositol, a-methyl-D-glucoside, Cellobiose, Maltose, Trehalose, Raffinose, Dextrose, Sodium sulfite, Incubator, Distilled water, Erlenmeyer flasks, Clothespin, 1% phosphomolybdic acid, Safranin, Glass slides & cover slips, 1% methyl green, Sabouraud dextrose broth, Sabouraud dextrose sugar, Glass slides and cover slips, Staining tray, Gum labels and permanent marking pens, Clean type, Clean nail polish, Petri dishes, 0.5 McFarland Standard, Yeast extract, Glucose, Agar, L-arabinose, Adonitol, Galactose, Sorbitol, N-acetyl-D-glucosamine, Lactose, Saccharose, Mellizitose, Glycine, Bismuth ammonium citrate

• Label slide at one end which is unlikely to contact the stain with Organism used, Staining method used, Name of student reminding of uses clean, grease free, dry slides

• To save space, abbreviations for organism and staining method may be used

• Use a sterile and cooled loop to transfer loopfuls onto the slide and spread out the smear on the blank portion of the slide

• Dry the smear in air and heat-fix by passing the slide film sideways three to five times over the flame, using a slide holder or clothespin

• Flame-sterilize the loop before and after using it

• Too much broth culture on the slide would delay the air-drying process with heat-fixing serving to make the cells adhere to the slide

• Prepare smears that are neither too thick nor too thin because thick smears will be unevenly stained and it will be tough to see individual cells

• Prepare a smear of 24-48- hour broth or agar culture of yeasts

• Cover the smear with crystal violet for 1 minute with a dropper

• Tilt the slide and pour off the stain into an empty beaker after 1 minute

• Rinse off the remaining stain using a wash bottle with the slide tilted

• Carefully blot-dry the smear with tissue paper and examine cells under high-power objectives (HPO) and oil-immersion objectives (O1O).

• Prepare yeast smears and perform cell wall staining and after Gram staining drying the slide and treat with 1% phosphomolybdic acid for 5 minutes

• Drain and rinse the slide with water, tap the excess water from the slide onto a piece of tissue paper

• Stain using a 1% aqueous methyl green for 30 seconds and after 1 minute, drain and rinse, blot-dry & examine under OIO noting that intact cells are dark purple or black, broken cell walls are light green, and exposed cytoplasm is light purple and to not overstain with timing very critical in Gram staining

• Follow the Determination of Oxygen Requirement protocol by inoculating the Sabouraud dextrose broth with the inoculum and mix

• Incubate for 48 hours at the optimum temperature of the organism and observe

• Use a cell viability dye as an indicator to determine if the cell is actively dividing in this case using a greenish-blue color to indicate viable cells when methylene blue dye is used as an indicator, or a pink color if resazurin dye is used as an indicator with the following characteristics:

  • Growth at the upper surface: obligate aerobes
  • Even growth throughout: aerotolerant anaerobes
  • Growth only at the bottom: obligate anaerobes
  • Growth throughout, but more growth at the upper surface: facultative anaerobes
  • Growth only near but not on the surface: microaerophiles

• Used to determine the ability of the yeast to use a particular sugar as its carbohydrate substrate and sole carbon source in the medium the Carbohydrate Assimilation Test

• The growth of yeasts, which is shown as the turbidity of the solution, denotes a positive result in this test

• In the yeast culture in a Sabouraud dextrose broth for 24 hours at 30 °C, after incubation, the culture will undergo centrifugation for 5 minutes at 2,700 x g with the pellets washed and centrifuge them three times, and then suspend the pellets with sterile saline solution, and adjust the turbidity to 0.5 McFarland standard

• Prepare tubes and place 2 mL of Yeast Nitrogen Base (YNB) media after which on each tube, add one specific carbohydrate (glucose, glycerol, 2-keto-D-gluconate, L-arabinose, D-xylose, adonitol, xylitol, galactose, inositol, sorbitol, a-methyl-D-glucoside, N-acetyl-D-glucosamine, cellibiose, lactose, maltose, saccharose, trehalose, melezitose, raffinose), then make the final concentration to 2%

• Incubate at 30°C for 96 hours observing and taking note of the results that turbidity of the solution indicating active cell division

• Used to determine specific genera and species of yeast that can resist the presence of copper in the medium with the Copper Resistance Test

• Copper catalyzes H2O2 to produce a powerful hydronyl radical (OH*) that causes cellular damage and disrupts cellular function stating that some species of yeast resist the presence of copper by producing superoxide dismutase and metallothionein

Preparing a YPG (Yeast extract, Peptone, Glycerol/Glucose) agar medium

• Consisting of 1% yeast extract, 2% peptone, 3% glycerol, or 2% glucose, and 2% agar dissolve the media using distilled water, then add copper sulfate to a final concentration of 62 uM, autoclave the prepared media and pour it into sterilized Petri dishes,
• Inoculate the yeast sample by streak plate method into the agar plates then incubate the culture plate at 30°C for 24 to 72 hours and observe the colony growth per day listing that BiGGY agar is a selective medium used to isolate and presumptively identify Candida species through their ability to reduce sulfite to sulfide, which gives the media a brown or black color which is prepared with a BiGGY (Bismuth, Glycine, Glucose, Yeast Extract) agar medium containing 1 g/L yeast extract, 10 g/L glycine, 10 g/L dextrose, 5 g/L bismuth ammonium citrate, 3 g/L sodium sulfite, and 16 g/L agar by dissolving the media in distilled water and mix thoroughly
• Heat the solution gently until the medium is completely dissolved
• Do NOT autoclave the media because it will destroy some of their components
• Dispense the media into the Petri dishes
• Inoculate the yeast sample by the streak plate method onto the agar plates and incubate the culture plate at 30°C for 48 hours and observe the colony growth and characteristics

Morphological & Biochemical Characterization of Molds

• Molds are a type of fungus that grows as multicellular filaments

• These multicellular filaments or threads are called hyphae (sing. hypha) and form a mycelium-branched network, which the hyphae are either vegetative or aerial

• Vegetative hyphae obtain nutrients from the source and grow laterally in the growth medium, and aerial hyphae rises and produces spores vertically from the growth medium

• Asexual spores are produced by sporangia (sing. sporangium) or conidia (sing. conidium), being asexual spores from conidia are called conidiospores

• Asexual spores enclosed in a sporangium are called sporangiospores

• Aerial hyphae containing conidia and sporangia are called conidiophores and sporangiophores, respectively

• Certain molds can survive in acidic conditions and in high concentrations of sugar or salt where they grow as parasites and saprophytes, causing considerable damage to food products

• Some molds are used commercially to produce cheese

• By visiting to a grocery store, several specimens of mold can be produced as many fruits and vegetables often develop mold during shipping listing examples such as Roquefort and Camembert cheeses rely on mold growth giving the cheese its distinct flavor with dark, fluffy lines showing where mold that can also be prominent in discarded parts of cheese or stale bread

Morphological Characterization

• The objectives are to describe the morphological features of molds, to classify species of molds using dichotomous and picture keys, and to screen sources of molds for identification Materials and Reagents include: Food sources with mold growths (e.g., bread, fruits, Lactophenol vegetables, cheese, etc.) Compound light Bunsen burner/alcohol microscope lamp Sterile tweezers/forceps Slides 7 cover slips Scotch tape Test tubes Petri dishes Distilled water Sabouraud dextrose broth Clear nail polish Potato dextrose agar (PDA) Sabouraud dextrose sugar Cellulose Malt extract sugar KH2PO4 Yeast extract CaCl2 dihydrate MgSO4 heptahydrate Agar Esculin Inositol Ferric sulfate Tryptone Xylan Skimmed milked powder Dextrose Gelatin NaCl Coomassie Brilliant Blue Methanol G-250 (NH4)2SO4 Acetic acid FeSO4 heptahydrate Na2HPO4 MnSO4 H3BO3 CuSO4 ZnSO4 Hexadecyltrimethyl MoO3 ammonium bromide Carboxylmethylcellulose Pectin Glucose Congo Red dye p-cresol (4-methoxyphenol) Glycine Bromocresol purple Cork borer Histidine Phenylalanine Lysine Tyrosine Inoculating needle Ornithine

Begin the Procedure – Morphological Characterization by describing its: - Colony Morphology; preparing tree-culture media (Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA), and Malt Extract Agar (MEA)) inoculating a pure culture of molds isolated from different sources onto the three-culture media and incubating at 30°C for 72 hours to observe the growth in the media - Describe the colony characteristics of the mold growing in the culture medium by its form, size, elevation, margin, surface and color or pigmentation starting with the labeling of slides before

• Prepare a slide labeling procedure starting from that the Label Slide slide at one end that is unlikely to contact the stain with the organism used, the staining method used, and the name of the relevant party while using clean, grease free and dry slides with space, abbreviations that can be used for the organism, as well as the stain method

• By mixing a small amount of growth with the drop of water on the slide adding a sterile and cooled loop and by spreading evenly with the loop as well as air-drying and heat-fixing; the loops need to be flame-sterilized before and after use

• Prepare a smear of 24-48- hour broth or agar culture of yeasts using a mixture including sterile loops, slides with organisms and growth with added solutions

• Used to determine the ability of the yeast to use a particular sugar as its carbon the Morphological slides needs used in cultures with sterile loops along with specific tests for each solution adding particular carbohydrates

• Labeling the slides, slide cultures, growth, tests and carbohydrates added, after which they are sterilized

• The objectives are to: describe the morphological features of molds; classify species of molds using dichotomous and picture keys; screen sources of molds for identification

• There are Colonial Characteristics of molds *with their *Margins with descriptions of the Entire, Undulate and Irregular, branching in Lobate or Ciliate patterns, from Wooly, Hair-lock like or Threadlike forms

• There are forms such as Round, Wrinkled, Filamentous or Complex structures with a Round with raised margin, Concentric Round with radiating margin or Filiform structures

• The three descriptions of structures being Round that are then made up of irregular or spread patterns making Rhiozoids with an L-form, while *Elevation with a Raised, Convex or Flat form Umbonate to Crateriform

• Describe the morphological features of molds

• There are specific genera and species with spore samples in sterile water

• The inoculum will be suspended in Sabouraud dextrose broth

• Use a cell viability to determine oxygen requirements by adding the sample and incubating, noting which of the growths are near, above or below based on those descriptions

• Beta-glucosidase catalyzes the hydrolysis of alkyl and aryl-beta glucosides in oligosaccharides leading some mold to produce this enzyme for hydrolysis

• Cellulase Activity is the method to degrade the growth of cellulose in Molds

Colonial Characteristics

• *Some molds produce this enzyme to hydrolyze and depolymerize cellulose through cellulasic activiity, into, , some monomers with that specific ability

• *With the tests listed, use water or tested chemicals in each culture, observe them, note them to determine which species or strain the mold is.

• Molds also produce acid and alkaline proteases along with chitin to grow better with that ability to resist pressure and resistance in harsh solutions

• Prepare a slide to view under microscope, observe and note the descriptions

• Use a Lactophenol in a pipette, with the sterilized mixture adding clear nail polish to seal the slide

• Observe the slides for patterns described and compared to standards, noting the results, to determine