MicroBioLEC_PRELIMS.pdf

BACTERIAL CELL STRUCTURE Viruses → parasitic → they do not replicate Three (3) Main Domains Prions and Virus 1. Bacteria → they are not cells (acellular) - fungi (multicel...

BACTERIAL CELL STRUCTURE Viruses → parasitic → they do not replicate Three (3) Main Domains Prions and Virus 1. Bacteria → they are not cells (acellular) - fungi (multicellular) → non-cellular infectious - mold (uni-) → agent/acellular microbes - yeast (multi-) → they do not have own metabolic machine - protozoa (uni-, parasitic) - algae (photosynthetic ability) PROKARYOTES 2. Archaea → LACKS: mitochondria, ER, Golgi 3. Eukaryotes apparatus → ABSENCE OF TRUE NUCLEUS Polymerase Chain Reaction (PCR) *Bacteria requires ATP but do not have Thermus aquaticus (TAQ) - reagent in mitochondria, instead in the cell PCR membrane ATP are generated* DNA Polymerase —> (38) generated ATP and glucose EUKARYOTES - enzyme produces DNA → humans: (36) generated ATP and - complex material glucose Polymers → polysaccharide (STARCH) MOLECULAR BIOLOGY → polypeptide (amino acid joined via peptide/amide) PCR → Copy DNA into millions → lipids Archaea → lives in coastal environment → polynucleotide (DNA/RNA) Thermophiles Microbial cells have polymers → can stand in high temp. - found in cytoplasm → πe 56 - 60° - Denaturation temp. - no nucleus DNAP TAQ polymerase → Thermus aquaticus - source of Thermus aquaticus - bacteria that enzyme ligase thermophiles can live in at high temp. Ligase: molecular glue joins together target DNA with Reverse transcriptase (RT-PCR) - a enzyme vector DNA. - RNA → DNA Pyrococcus furiosus - example of SARS-COV - RNA virus (viruses carries DNAP-PCR only one polypeptide) Restriction enzymes - molecular scissors  Bacteria that are used as Sources of → Alexander Fleming Penicillin Restriction Enzymes (Penicillium notatum) HindIII - from Haemophilus influenzae Biodegration → fungi BamHI - from Bacillus amyloliquifaciens mix culture (fungi & bacteria) Haelll - from Haemophilus aegyptius Cytoplasm → nucleoids Alul- from Arthrobacter luteus Double circular (No Histone) EcoRI - from Escherichia coli → Histone octamen BACTERIA no membrane; (ATP ) - cell Food industry membrane Laboratory → Lactic acid bacteria (most on the Ribosomes (protein synthesis) leaves) —> Svedverg = "s" → product of fermentation - lactic acid Eukaryote 80s (40s small, 60s large) Corynebacterium glutamicus - MSG → harmful (in humans) Prokaryote 70s (50s large, 30s small) C. diphtheriae (deadly) → super infectious *fungi has cell wall → pseudomembrane in throat *bacteria has cell wall (peptidoglycan) *fungi has cell wall (chitin) C2420H - acetaldehyde *protozoa has no cell wall -> Lower alc. dehydrogenae - (Female) -> Higher alc. dehydrogenae - (male) BACTERIA CELL WALL (Peptidoglycan/murein) alcohol -> acetaldehyde -> acetic acid → Gram positive (thicker peptidoglycan) Primary metabolites → Gram negative (thinner peptidoglycan) → metabolism, growth and reproduction → end product of metabolism saliva - rich in lysozyme (tears & saliva) (e.g., alcohol, glutamate, a.a) that digests peptidoglycan → during the logarithmic phase, when *antibiotic killed peptidoglycan* bacteria actively dividing primary (e.g., betalactam antibiotics) metabolites activate. Properties Gram (+) Gram (-) Secondary metabolites Cell wall thicker (20-25 nm) Thinner (11-15 → product of primary metabolites nm) Gram deep blue pink -> red → produce during the stationary phase of reaction (violet/purple) bacterial growth LPS ABSENT PRESENT (e.g., antibiotics) (bacteriocin) (outer Bacteriocin: role is to kill other bacteria membrane) Peptidoglyc PRESENT (multi; PRESENT Pili - For attachment to an layer 50% more) single-layered thin (10%) Structures sometimes present Cell wall - containing murein, a Periplasmic ABSENT PRESENT layer (bet… murein & peptidoglycan inner Cell surface membrane bet… murein & Cytoplasm outer) Circular DNA - sometimes referred Flagellar 2 rings basal body 4 rings basal body to as a chromosome Ribosomes Lipid Low High content NAM-NAG (sequence of DNA in bacteria) Lysozome Protoplast Sphoroplast (NAM - N-acetyl muramic acid) (easily destroyed) (refractory) (NAG - N-acetyl glucosamine) e.g. C spp., B spp., Escherichia coli, Listeria Klebsiella Mycobacterium tuberculosis monocytogenes, pneumoniae, (acid fast bacilli) Staphylococcus, Pseudomonas → Mycolic acid or hydroxymethoxy acid Streptococcus, aeruginosa, -> a major component of M. tb cell wall etc. Neiserria gonorrhoeae, → wax/ lipid (resists staining) Vibrio cholerae ACID FAST STAINING TECH. Protoplasts: Cells whose walls have been (Ziehl Neelsen) completely removed are incapable of normal growth and division. Initial stain - Carbol Fuchsin (phenol added in the preparation of carbol fuchsin) Spheroplasts: Gram-negative bacteria with Mordant - Steam the intact cytoplasmic membrane of the Decolorizer - Acid alcohol protoplast plus the outer membrane (LPS Counter stain - Malachite Green/Methylene layer) of the cell wall, after the Blue peptidoglycan layer is destroyed by lysozyme or its synthesis inhibited by INTERPRETATION: antibiotics. AFB = red bacilli NAFB = blue/green Additional structures sometimes present Flagellum - For locomotion; very SPUTUM: specimen of choice in diagnosing simple structure pulmonary tuberculosis Capsule - additional protection Infolding of cell surface ENDOTOXINS membrane - may form a → produced gram negative (-) photosynthetic membrane or carry → associated within inflammatory action but nitrogen fixation plasmid → produces when bacteria lies Plasmid - small circle of DNA: → components of the outer membrane several may be present  CAPSULE (repeats of polysaccharides) Virulence factor TYPES OF ANTRAX → degree of pathogenicity a. cutaneous Pathogenicity → from sheep (black escher) → ability to cause diseases b. Respiratory → terrorist Streptococcus pneumoniae/Diplococcus c. Gastrointestinal pneumoniae (gram (+) cocci) → deadliest antrax → causes diarrhea and vomiting Non - virulence: Rough stain: (through ingestion) non-encapsulated → young lamb Virulence: Soft stain: encapsulated 3. B. cereus Neisseria meningitidis - causing meningitis → fried-rice food poisoning (SIALIC ACID) Clostridium spp. Streptococcus pyogenes belonging Grp A = (HYALURONIC ACID) 1. C. perfringens Sterile → Gas gangrene (anaerobic bacilli) → zero (0) microorganisms 2. C. botulinum → cerebrospinal fluid → canned- good bacillli → blood (SEPSIS) Sepsicemia 3. C. tetani ENDOSPORES (calcium dipicolinate) TRIGGER TO ENDOSPORE FORMATION → inside the cell Calcium dipicolinate - chemical Vegetative Cell (non-spore forming cell) composition of endospores (through nutrient deprivation; aging of culture; starvation; harsh environmental condition (desiccation)) → Present in Bacillus and Clostridium Spore-Forming Cell Bacillus spp. Powdered bacteria 1. B. subtilis → common laboratory contaminant Flagella → Motility → source of bacitracin (bacteriocin) Atrichous - no flagella; immotile 2. B. anthracis Monotrichous- one flagellum → common bioterrorism agent Peritrichous - numerous flagella → largest pathogenic bacteria of man surrounding the cell (most motile) → gram (+) rod Ex. Escherichia coli - a normal gut → ANTRAX (diseases) microbial flora Lopotrichous - a tuft of flagella on one pole.  Amphitrichous - flagella on both poles. Turbidity on medium = (+) results PLASMIDS Slow growers (Tubercle bacilli) 20 hours → Extrachromosomal DNAs that may be Mycobacterium tuberculosis present in some bacterial cells. → chronic infection → Double-stranded, circular, → early morning sputum for sample covalently-bound DNA. → 3 successive negative results / days for *Genes in the plasmids do not play vital clearance functions to cells.* CAUSES: TYPES OF PLASMIDS → low-grade fever esp. Afternoon → loss of appetite 1. F plasmid = codes for pilus/pili → sudden weight loss → extracellular appendage wherein → cough for more than 3 weeks bacteria are passed from one to another. Dye Classification 2. R plasmid = codes for drug resistance a. Positive → carries antibiotic resistance gene → stains the cell/tissue (ARG) → presence of negative sialic acid e.g, MRSA - Methicillin resistant → attracted to a negative cell surface staphylococcus aureus e.g. methylene blue, malachite green, crystal violet, safranin HOW DO BACTERIA REPRODUCE? → By ASEXUAL REPRODUCTION 1. Simple - 1 dye (binary fission - cell divides every 2. Differential - 2 or more dyes certain time) → Gram staining technique → hematoxylin and eosin Stages in Bacterial Growth Note: 1. lag phase b. Negative 2. log phase → stains background due to → acclimatization/period of adjustment repulsion, thus cells are colorless → new environment = culture media e.g. India ink, nigrossin 3. stationary/plateau phase 4. death/decline phase detects fungal meningitis (Cryptococcus neoformans) Incubation Growing bacteria in incubator is: detects bacterial meningitis (Haemophilus 35-37°C for 18-24 hours OR 24°C for influenzae) more than 3 days → major cause of meningitis in newborn → specimen determines meningitidis - hazy Usual generation time of ordinary CSF and treated via lumbar tap. non-fastidious bacteria is 20 mins. Gram Negative Staining Motile Bacteria (monotrichous, peritrichous, lopotrichous, 1. Crystal Violet - initial stain amphitrichous) 2. Gram’s Iodine - mordant mordant - color enhancer/intensifier; Vibrio cholerae - monotrichous increases affinity of dye to cell Escherichia coli - peritrichous 3. Ethyl Alcohol - decolorizer Shigella dysenteriae - atrichous 4. Safranin - counter stain Non-motile V→I→A→S (ex. Shigella dysenteriae- causes bacillary dysentery) - ATRICHOUS Describing a Bacteria 1. Gram Stain reaction (+) and (-) Molecular Characterization 2. Cell shape Ex. -> cocci 16S rRNA gene sequencing (bacteria) or -> bacilli 18S rRNA gene sequencing (eukaryotes) -> spirilla 3. Cell arrangement S for “Svedberg unit” (sedimentation -> diplo (pair) constant) -> tetrad -> cluster G:C Ratio (Guanine: Cytosine Ratio) -> sarcina (8) -> filament Physiological Characterization -> singly 4. Spores Energy Source (phototroph or -> central chemotroph) -> terminal -> subterminal Carbon Source -> heterotroph - uses sugar for carbon Phenotypic Characterizations -> lithotroph - uses inorganics for electron and energy (e.g., iron bacteria in red, 1. Gram staining reaction ferrous soil) (Gram positive vs Gram negative bacteria) Physiological Needs of Bacteria Gram positive (+) bacteria - purple Oxygen Requirements Gram negative (-) bacteria - red or pink *In humans, too much Oxygen causes blindness.* 2. Bacterial shape (cocci, bacilli, spirilli) Bacillus vs bacillus Obligate aerobes → has catalase and peroxidase that 3. Cell arrangement (in pairs, in tetrad, → use cotton plug grapelike cluster, chain-like arrangement) e.g. all Bacillus (a.) B. subtilis (b.) B. anthracis 4. Motility (motile or non-motile) (c.) B. cereus (d.) B. thuringiensis –  anti-pest (ex. pyogens - bacteria that can withstand Obligate anaerobes very high temp., Pyrococcus furiosus, → NO catalase and peroxidase Thermus aquaticus) → sediments growing in broth culture → use cork for closure EXTREMOPHILES e.g.: (ex. Archaebacteria; Thermus aquaticus); C. perfringens – gangrenous legs of Archaebacteria vs Eubacteria diabetic patients which are treated by hyperbaric oxygen pH Requirement C. tetani – tennis racket bacilli that cause ACIDOPHILES- prefer an acidic tetanus/lockjaw environment/ low pH (Ex. Lactobacilli spp. C. botulinum – canned good bacilli that (Lactic Acid Bacteria) cause botox C. difficile – cause of pseudomembranous Cariogenic bacteria - they can cause tooth colitis, which is treated by Clindamycin. decay or dental caries Fungi are acidophiles (Candida albicans - Facultative anaerobes – aerobes that adapt yeast that may cause vaginal thrush) in few oxygen environments e.g. coliform/enteric/colon bacteria ALKALINOPHILES - prefer high pH → Escherichia coli – greenish metallic (alkaline) on agar plate Ex. Vibrio spp. Uses TCBS culture medium → Salmonella typhi (thiosulfate Citrate Bile Salt Sucrose) - → Enterobacter aerogenes alkaline pH (pH= 8.6) Microaerophiles – can live in small amounts of Oxygen Vibrio parahemolyticus - shellfish food e.g. Helicobacter pylori and Campylobacter poisoning jejuni Vibrio cholerae - "rice watery stool cholera" Vibrio fischerii Temperature Requirements CO2 REQUIREMENT MESOPHILES - 30-40C CAPNOPHILES - prefer increased CO2 ave: 35 - 37 C Human body temperature) - tension human pathogens (ex. Salmonella typhi - Disease causing Streptococcus pneumoniae (a fastidious (pathogenic) bacteria that causes deadly lobar pneumonia) PSYCHROPHILES - cold-loving bacteria Helicobacter pylori - bacteria that causes (10-20 C) ulcer (ex. Listeria monocytogenes - multiple in Campylobacter jejuni - bacteria that causes refrigerator) Gastroenteritis THERMOPHILES - 100-600 C OSMOPHILES - can tolerate high osmotic medium  PLASMOLYSIS - cell shrinks once Colonial Characterization/ Colonial exposed to a hypertonic environment Morphology PLASMOPTYSIS - Swelling of the cell as Colony size (pinpoint, pinhead, small, a result of water from the hypotonic medium or large colonies) medium entering the cell Color of the colonies (buff, yellow, white, HALOPHILES - prefer high salinity green) medium (10% NaCl) Pseudomonas aeruginosa - blue green Ex. Vibrio spp. water-soluble pigment (pyoverdin, pyocyanin - produced by Biochemical Tests pseudomonas aeruginosa) Gram Positive Cocci pyoverdin - green Catalase and Coagulase Tests pyocyanin - blue CATALASE TEST - presumptive test for Elevation (flat, raised, concave, convex) differentiating Staphylococci from Streptococci BIOPIGMENTS Positive Result: bubble formation Serratia marcescens H2O2 —> catalase---→H2O + O - red pigmented Colonies (water insoluble) Staphylococci are catalase positive - PRODIGIOSIN Streptococci are catalase negative Gram Negative Bacilli COAGULASE TEST - differentiates (Family Enterobacteriaceae) pathogenic Staphylococci from Non-pathogenic one I - Indole Test (NEGATIVE) RED Staphylococcus aureus - agent of acne, M - Methyl Red (NEGATIVE) RED pimple, folliculitis, carbuncle, furuncle, boils and food poisoning (Coagulase positive) V - Vogues Prauskauer Test (NEGATIVE) RED Staphylococcus epidermidis - occasionally may cause STITCH C - Citrate Test - (POSITIVE) ABSCESS; normal flora of the skin BLUE/GREEN (Coagulase negative) MRSA - Methicillin Resistant Staphylococcus aureus METHICILLIN - Betalactam (resistant to betalactam antibiotics INDOLE TEST METHYL RED TEST Principle: Detects the ability of coliform to Principle: used to determine if bacteria produce TRYPTOPHANASE which can can perform mixed acid fermentation. hydrolyze tryptophan to pyruvic acid, uses MRVP broth ammonia and INDOLE. INDICATOR OF PH if methyl red is red, it is acid Ehrlich's reagent/Kovach’s (indicator) = if methyl red is yellow, it is alkaline 0.5 mL permenter - automatic produce acid (byproduct) (paradimethylaminobenzaldehyde) = red when indole is present Methyl Red Test para-Dimethylaminobenzaldehyde is an Methyl Red detects acidity and indicator of organic compound containing amine and pH (low pH) in the culture medium aldehyde moieties which is used in Ehrlich's reagent and Kovac's reagent to - If the medium is highly acidic, it results in test for indoles. red coloration; if the medium is less acidic, it results in yellow coloration. (A) Escherichia coli (Proteus vulgaris) Indole positive pH is > 4.4 = red (+ result) (When the pH of the culture medium (B) Klebsiella pneumoniae (Entrobacter becomes pH 4.4 or less, a red spp.) Indole negative color is seen as positive result) Indole Test Culture Medium: MRVP medium (broth) - Detects the enzyme tryptophanase Glucose (substrate) + bacteria produces red coloration (fermenters) → acid products Culture Medium: Clark Lubs tryptophan broth medium Reagent: methyl red (pH indicator) Substrate: tryptophan (AA) (deamination) N Positive Bacteria: E. coli Product: Indole pyruvic acid Reagent: to detect the presence of Indole, TRIPLE SUCROSE IRON AGAR add Ehrlich reagent/Kovach's reagent: (slant butt agar) paradimethyl aminobenzaldehyde (Positive Result: red ring) USE: differentiates enterics based on Positive Bacteria: Escherichia coli; Proteus their ability to ferment carbohydrates and spp. reduce sulfur. The TSI medium contains: three carbohydrates--glucose, lactose, and sucrose-- and iron ions, sodium thiosulfate, and the pH indicator phenol red. The medium is usually made as a 'slant' agar in a glass tube.. VOGUES PRAUSKAUER TEST determines whether the given bacteria produces Acetoin (acetylmethylcarbinol) CITRATE UTILIZATION or not PRINCIPLE: detects production of the If an aerobic bacteria produces acetoin, enzyme, CITRATE PERMEASE (CITRASE) it reacts with added alpha napthol in which converts citrate to pyruvate; presence of strong alkali to produce determines the ability of bacteria to utilize diacetyl. Diacetyl further reacts with sodium citrate as its only carbon source, guanidine groups of proteins (peptone) and inorganic ammonium dihydrogen to form red or pinkish color product. phosphate as the sole nitrogen source CULTURE MEDIUM: Simmon Citrate Agar INDICATOR: Bromthymol Blue determine if an organism produces (changes color from green to blue at a pH (acetylmethylcarbinol) from glucose above 7.6) fermentation. Positive Reaction: Growth with color From beige yellow to a pink-red shade change from green to intense blue along the slant. Vogues Prauskauer Test Examples: Salmonella, Edwardsiella, Vogues Prauskauer Test is based on the Citrobacter, Klebsiella, Enterobacter, ability of bacteria to produce ACETOIN from Serratia, Providencia, etc. CHO fermentation. - bacteria utilize glucose in the process of Negative Reaction: No growth and No glucose utilization, acetoin is produced. color change; Slant remains green. Culture Medium: MRVP broth medium Reagents: Examples: Escherichia, Shigella, Barrit's A-alpha naphtol Morganella, Yersinia, etc. Barrit's B -40% KOH (results: intense pink coloration) Citrate Test Positive Bacteria: Citrate Test is based on the ability of Enterobacter aerogenes (coliform) bacteria to use citrate as sole carbon source, they produce alkaline products  GREEN TO PRUSSIAN BLUE A/A = (lactose fermenter) - If the color remains green, (-) result lactose fermenter grp. obtained Escherichia coli - If the bacteria is inoculated in Simon Enterobacter aerogenes Citrate agar, color turns Prussian Blue (+) Klebsiella pneumoniae result Culture Medium: Simmon Citrate Agar - Medium is displaced, bubble, or (SCA) cracked; the agar will push up, meaning it Indicator: Bromthymol blue have gas production Positive Result: royal or Prussian Blue (A/A + GAS(+) OR A/A G(+) - Escherichia Positive: Enterobacter aerogenes coli) Negative: Escherichia coli confirmatory test of E. coli - A/A G(+) Escherichia coli IMVIC Test Results: red(+), red(+), RED SLANT and YELLOW BUTT = K/A yellow(-), green(-) K/A = (non-lactose fermenter) Enterobacter aerogenes K/A = pathogenic bacteria, Salmonella IMVIC Test Results: yellow(-), yellow(-), (agent for typhoid fever) and Shigella red(+), blue(+) Blackening at the bottom, blackening Triple Sugar Iron MEDIUM (TSI) (H2S) Hydrogen Sulfide - indicated by black color Content of TSI: three sugars (Glucose, K/A+ H2S and K/K + H2S Lactose & Sucrose If the bacteria is lactose fermenter, the Indicator: PHENOL RED lactose sugar is fermented Acid: yellow; Alkaline: red lactose sugar, the glucose sugar is Ferrous ammonium sulfate as indicator of as well fermented Hydrogen sulfide production If two (2) sugar (glucose and lactose) or Gas production in the form of: (1) bubble; more sugar are fermented, then TSI is (2) displacement of the slant/butt and (3) YELLOW SLANT and YELLOW BUTT crack in the medium (A/A). Blackening: production of hydrogen sulfide LF: E. col; E. aerogenes: K. pneumoniae If only one (1) sugar is fermented, that sugar is glucose, it results RED SLANT and (PINKISH RED - uninoculated/ no broth) YELLOW BUTT (K/A). Method of inoculation: stob and strike; butt slant), incubation: 18 - 14 hrs If none of the three (3) sugars is fermented, it results (K/K) and the entire If the medium becomes YELLOW SLANT tube is red. and YELLOW BUTT, (A/A) *If the lactose fermented along with glucose, *If the bacteria is lactose fermenter, one of the bacteria is lactose fermenter.* the products produced acid, from an indicator of neutral red into pink. If alkaline, lactose fermenter grp. the product is yellow.* Escherichia coli Enterobacter aerogenes TSI/McConkey Klebsiella pneumoniae Escherichia coli = RED/PINK = Lactose fermenting colonies A/A = Escherichia coli, Enterobacter Enterobacter aerogenes = aerogenes, Klebsiella pneumoniae RED/PINK K/A = pathogenic bacteria, Salmonella Proteus vulgaris - causes UTI (agent for typhoid fever) and Shigella (agent for bacillary dysentery) If TSI is K/A, McConkey colorless colonies = non-fermenting colonies K/K = Pseudomonas Escherichia coli in Eosin Methylene Blue RESULTS OF TMI AND IMVIC USING (EMB) COLONIAL MORPHOLOGY uninoculated medium: maroon color Cultural characterization: - EMB is also selective and differential CM Gram (-) bacilli rod (belonging in Family *Selective because dyes such as Enterobacteriaceae), it must grow in eosin and methylene blue to inhibits the McConkey agar growth of gram (+) bacteria* *only gram (-) bacteria will grown McConkey agar - a selective and in EMB agar* Differential medium (CM); contains * E. coli in EMB agar inhibits inhibitors (bile salts and crystal violet) quaternary strikes* that prevent gram (+) bacteria from growing *greenish metallic sheen indicate in this media. the presence of E. coli in EMB agar, SELECTIVE MEDIUM - selects only fish-eye isolated colonies* gram (-) bacilli pH indicator: Neutral red *always on last last strikes can able to have isolated colonies that will use in biochemical McConkey agar contains lactose; tests.* Lactose = sugar in McConkey agar serves as energy in bacteria TYPES OF CULTURE MEDIA *When testing biochemical tests, tertiary Primary Isolation - Simple Culture strikes have isolated colonies that are Medium (NB; NA) possible in TSI and IMViC.*  Enriched/Enrichment - added nutrients in Special: PDA (Potato Dextrose Agar/ Culture Medium; for isolation of fastidious Dextrose Agar) = fungal isolation bacteria (Ex. Brain Heart Infusion Agar and Vibrio spp. = TCBS Thiosulfate Citrate Bile Broth (BHIA; BHIB)) Salt Sucrose Agar Selective - inhibitors are added to select the growth of the desired bacteria; (McConkey Agar and EMB) Differential - differentiate colonies (whether LF or NLF); differentiate hemolytic patterns of bacteria (alpha, beta and gamma hemolytic) (Ex. Blood Agar Plate (BAP); McConkey Agar and EMB) Hemolysis - blood breakdown → Alpha-Hemolysis(α-hemolysis) agar under the colony is light and greenish. This is sometimes called green hemolysis because of the color change in the agar. e.g. Streptococcus pneumoniae and a group of oral streptococci → Beta-hemolysis (β-hemolysis) called complete hemolysis, RBC cells in the media around and under the colonies: the area appears lightened (yellow) and transparent. e.g. Streptococcus pyogenes, Staphylococcus aureus → Gamma-hemolysis (γ-hemolysis) the organism does not induce hemolysis, the agar under and around the colony is unchanged. e.g. Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus epidermidis.