Principles of Different Microbial Tests PDF

Republic of the Philippines CENTRAL BICOL STATE UNIVERSITY OF AGRICULTURE Calabanga | Pasacao | Pili | Sipocot Department of Food Science FST 101 – Food Microbiology PRINCIPLES OF GRAM STAINING, ENDOSPORE FORMATION...

Republic of the Philippines CENTRAL BICOL STATE UNIVERSITY OF AGRICULTURE Calabanga | Pasacao | Pili | Sipocot Department of Food Science FST 101 – Food Microbiology PRINCIPLES OF GRAM STAINING, ENDOSPORE FORMATION, CATALASE TEST, MOTILITY and FERMENTATION TEST ALESSANDRA M. DOMANACO Assistant Professor IV GRAM STAINING Gram-Positive Bacteria Gram-Negative Bacteria  OVERVIEW OF GRAM STAINING ▪ Originally developed by Hans Christian Gram (1853) - Danish physician - Studied botany at the University of Copenhagen in Denmark. - Discovered that certain stains were preferentially taken up and retained by bacterial cells. - His initial work with staining process was performed on Streptococcus pneumoniae and Klebsiella pneumoniae. ▪ Carl Weigert (1845 – 1904) - German pathologist - Added a final step of staining with safranin (counterstain).  The staining technique that Gram developed is still the most important method for distinguishing between two major classes of bacteria. GRAM STAINING PROCEDURE PREPARATION OF SMEAR STAINING PROCEDURE RESULTS: MICROSCOPIC VIEW Gram positive bacteria Gram negative bacteria RESULTS: MICROSCOPIC VIEW Mix: Gram negative & gram negative bacteria RELATIONSHIP OF CELL WALL STRUCTURE TO THE GRAM STAIN What makes some cells retain crystal violet (Gram positive) while others readily release the stain when alcohol is added (Gram negative)? Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50- 90% of cell wall), which stain purple and Gram-negative bacteria have a thinner layer (10% of cell wall), which stain pink. Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. In the Gram stain, an insoluble crystal violet-iodine complex is formed inside the cell, and this complex is extracted by alcohol from gram-negative but not from gram-positive Bacteria. The alcohol dehydrates Gram-positive Bacteria, which have very thick cell walls consisting of several layers of peptidoglycan. This causes the pores in the walls to close, preventing the insoluble crystal violet-iodine complex from escaping. In gram-negative Bacteria, alcohol readily penetrates the lipid-rich outer layer, and the thin peptidoglycan layer also does not prevent solvent passage, thus, the crystal violet-iodine complex is easily removed.  Result of gram staining: Blue to purple Bacillus subtilis (Gram – positive) Gram – positive cell wall (Gram – positive) Escherichia coli (Gram – negative) Gram – negative cell wall Result of gram staining: Pink to red (Gram – negative) ENDOSPORE FORMATION Endospores are formed generally in response to environmental conditions that are unfavorable for the continued growth of vegetative cells. The most common stimulus in nature is probably the exhaustion of nutrients. Germination of endospore: Bacterial endospores germinate to vegetative cells when placed in favorable condition, but most endospores cannot germinate after they have formed. They either need to remain dormant for some time or get activated before germination. The processes involved in germination of endospores are: 1. Activation 2. initiation 3. outgrowth 1.Activation: The endospore is activated in a nutritionally rich medium. The spore coat must be damaged for which agents like heat, abrasion, acid and compounds containing free sulf-hydryl groups are used. 2.Initiation: initiate germination under favorable environmental conditions. triggered by binding of endospore receptors with the effector molecules in the medium Binding of effector activates autolysin that rapidly degrades the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes. 3.Outgrowth: Uptake of water makes the endospore swell. A period of active biosynthesis follows, where DNA, RNA and proteins are synthesized. A new germ cell emerges out breaking the spore coat and becomes a vegetative cell. The vegetative cell grows and finally terminates in cell division with continuous supply of essential ENDOSPORE FORMER CATALASE TEST  Catalase test is used to demonstrate the presence of the enzyme catalase (catalase positive and catalase negative). Also valuable in differentiating aerobic and obligate anaerobic bacteria. Catalase enzyme - Is a common enzyme that is found in all living beings that survive in oxygen and catalyzes the decomposition of hydrogen peroxide, releasing water and oxygen. - Essential enzyme in pathogenic microorganisms as it protects them from oxidative damage from the reactive oxygen species.  PRINCIPLE OF CATALASE TEST ✓ The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water. ✓ The presence of the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen peroxide, and the rapid elaboration of oxygen bubbles occurs (Catalase positive) ✓ The lack of catalase is evident by a lack of or weak bubble production (Catalase negative) ✓ The culture should not be more than 24 hours old. ✓ Under the aerobic condition, 3% H2O2 is used, whereas 15% H2O2 is used under anaerobic conditions.  RESULT INTERPRETATION OF CATALASE TEST Positive: Copious bubbles produced, active bubbling Examples: Staphylococci, Micrococci, Listeria, Corynebacterium diphtheriae, Burkholderia cepacia, Nocardia, the family Enterobacteriaceae (Citrobacter, E. coli, Enterobacter, Klebsiella, Shigella, Yersinia, Proteus, Salmonella, Serratia), Pseudomonas, Mycobacterium tuberculosis, Aspergillus, Cryptococcus, and Rhodococcus equi. RESULT INTERPRETATION OF CATALASE TEST Negative: No or very few bubbles produced. Examples: Streptococcus and Enterococcus spp MOTILITY ✓ Motility is the ability of an organism to move by itself by means of propeller-like flagella unique to bacteria or by special fibrils that produce a gliding form of motility. ✓ Motile bacteria move using flagella, thread-like locomotor appendages extending outward from the plasma membrane and cell wall either single flagellum or multiple flagella. ✓ Each flagellum has a very rigid, helical structure, and actual motility results from the rotation of the flagellum in a manner similar to that of a boat propeller. ✓ Motility by the bacterium is demonstrated in a semi-solid agar medium. Positive: Escherichia coli, Enterobacteriaceae, Bacillus spp., Enterococcus, Yersinia enterocolitica Negative: Staphylococcus aureus, Acinetobacter LACTOSE FERMENTATION TESTS ▪ The ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds is a widely used method for the identification of microorganisms. ▪ Different fermentation media are used to differentiate organisms based on their ability to ferment carbohydrates incorporated into the basal medium. ▪ Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. ▪ Fermentation patterns can be used to differentiate among bacterial groups or species: ❑ e.g., ✓ all members of the Enterobacteriaceae family are classified as glucose fermenters. ✓ Within this family however, maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis (negative).  What is the purpose of the test? The purpose is to see if the microbe can ferment the carbohydrate (sugar) lactose as a carbon source. How is lactose fermentation determined? If lactose is fermented to produce acid end products, the pH of the medium will drop. A pH indicator in the medium changes color to indicate acid production. Requirements for Carbohydrate Test Culture media: Carbohydrate Broth Indicators: Andrade’s Indicator: light pink at about neutral pH range (7.1 to 7.2), turns dark pink to red at acidic pH (at around or below 5.0 ), and turns yellow at high alkaline pH (about 12 to 14) Phenol Red: Reddish orange at neutral (about 7.4 pH), turns yellow at acidic pH (at and below 6.8 pH), and turns pink-red at alkaline pH (at or above 8.4 pH) Bromocresol Purple: Deep purple at about neutral (about 7.4 pH), turns yellow at acidic pH (at and below 5.2 pH), and turns purple at alkaline pH (at and above 6.8 pH) Bromothymol Blue: Green at neutral pH (about 7.0 pH), turns yellow at acidic pH (at and below 6.0 pH), and turns Prussian blue at alkaline pH (at and above 8.4 pH) ▪ To detect these gases, a Durham tube is used. - This is a small inverted glass tube that is placed within the larger glass tube containing the fermentation medium. - If gases (typically CO2) are produced during the fermentation process, a bubble will form at the top of the Durham tube (meaning fermentation occurred).  ASSIGNMENT: METHYL RED AND VOGES – PROSKAUER (MRVP) TEST  ASSIGNMENT: Bacteria are identified and classified on the basis of their gram reaction, endospore formation, morphology and other distinguishing features. In line with this, differentiate from each other the given genera of bacteria. Construct a dichotomous key diagram to easily trace the similarities or differences of one genus from the other. Bacillus, Salmonella, Escherichia, Clostridium, Pseudomonas, Leuconostoc, Pediococcus, Staphylococcus and Enterobacter THANK YOU!