gp.1b. Structures, Taxonomy and Staining 18.pptx

Structures, Staining and Taxonomy Lecture 2 Glenn Patriquin MD  Structures of Bacteria (from outside in) Appendages – flagella – fimbriae – pili Surface and cell wall – capsule – cell wall – cell membrane Cytoplasm –...

Structures, Staining and Taxonomy Lecture 2 Glenn Patriquin MD  Structures of Bacteria (from outside in) Appendages – flagella – fimbriae – pili Surface and cell wall – capsule – cell wall – cell membrane Cytoplasm – chromosome – plasmids – ribosomes – inclusions Other structures – endospores https://micro.magnet.fsu.edu/cells/bacteriacell.html  Appendages - project from the cell Flagella – Long slender, structures made of protein – Whip like structures – Enable bacteria to move by rotating like a propeller – Can only be seen using special stains or electron microscopy – Can be single (monotrichous), or multiple, in tufts or around the cell (peritrichous).  Pseudomonas aeruginosa Flagella - Arrangements Bauman. Microbiology with Diseases by Body System, 5e  Appendages - project from the cell (cont’d) Fimbriae – Shorter, thinner filaments made of protein – Enable bacteria to attach to substances Pili – Similar to fimbriae in structure – Involved in transfer of DNA between bacteria Escherichia coli Proteus vulgaris Bauman. Microbiology with Diseases by Body System, 5e  Surface and Cell Wall Capsule – Material that is secreted by bacteria and covers the exterior of the cell – Often polysaccharide – May be a thick layer; slime coating Cell Wall – Differs from animal cells, or fungi – A strong layer made of peptidoglycan – Maintains cell shape and integrity – A principle target for antibiotic action – Stains using the Gram stain – Differs for Gram positive vs Gram negative organisms Peptidoglycan layer - Note cross links vertically and horizontally - NAG and NAM are chemical components that polymerize to form peptidoglycan - Gives a very strong lattice  Gram Positive and Gram Negative Gram Positive Gram Negative – Thick peptidoglycan – Outer membrane layer – Thin peptidoglycan layer – No outer membrane – Space between membranes is periplasmic space Gram Positive Gram Negative Acid Fast  Staining Organisms Unstained organisms can be seen using a “wet prep”- a suspension under a cover slip Stains are needed to allow us to see the organisms best using light microscopy Organisms are killed in the process Simple stains – stain is applied and colours the organism e.g. methylene blue https://microbe-canvas.com/tests.php?p=2400  Complex Stains stains may be combined to stain different structures different colours. e.g. giemsa stains malarial parasites nucleus red and cytoplasm blue stains may be applied in sequence with a step to remove stain in between. e.g. gram stain - a key stain in microbiology!!  The Gram Stain Developed by Christian Gram in the 19th Century He found that a stain could be washed out of some organisms much more easily than others Technique allows differentiation of many bacteria into 2 groups: gram positive and gram negative – corresponding to cell wall type. Continues to be used extensively and is important!  Method for Gram Stain Crystal violet – stains all the bacteria dark purple Iodine – binds to crystal violet and fixes it (acts as a mordant) Alcohol/Acetone* washes out the stain from gram negative bacteria Safranin stains the gram negative bacteria pink. *Gram originally stopped here, so that organisms that stained purple were “positive” because they could be seen; subsequently the fourth step was added so that both the positive and the negative organisms could be seen  The Gram Stain Gram Positive Gram Negative Before staining After crystal violet (primary stain) After iodine (mordant) After acetone/alcohol (decolourizer) After safranin (counter-stain)  Acid Fast Stain Some bacteria cannot be stained by the Gram stain because of lipids in the cell walls. (e.g. Mycobacterium tuberculosis, the tuberculosis bacterium) These bacteria may be stained by an “acid fast method”. Involves: – staining with a strong red stain (to “force” the stain into the cell) – washing out the stain with a mixture of acid and alcohol – restaining (“counterstaining”) with a blue or green stain. Acid Fast organisms are RED. These are sometimes called AFB (acid fast bacilli). Other organisms are the colour of the counter stain (blue or green). Cell Membrane Cell Membrane – Lipid bilayer with proteins – Controls the entrance and exit of substances from the cell – Contains enzymes involved in cell wall production, cellular metabolism, and production of some extra-cellular materials – In gram negatives, it contains endotoxin Cytoplasm – Liquid containing a variety of substances – Where metabolism occurs  Inside the Cell Ribosomes – Made of RNA and protein – Structures where proteins are made – Two subunits. Bacterial ribosomes are different from ribosomes in animal or plant cells (eukaryotic cells) Bacterial Chromosome – Made of DNA – A single long circular molecular of DNA – Not separated from cytoplasm (as in animal or plant cells which have nuclei) No Nucleus: Bacteria are Prokaryotic  Plasmids Plasmids – Small, circular pieces of DNA – Separate from the chromosome – Can be transferred between bacteria – May carry genes for antibiotic resistance Inclusions – Granules in the cytoplasm – May act as storage of various substances  Other Structures Endospores ("spore") – Environmentally tough, dormant form – Develop in cytoplasm of bacteria – Do not grow or divide – Can remain viable for long periods – Only formed by certain genera of bacteria – Germinates to form a new cell  Bacterial Taxonomy How are bacteria organized and classified – Domains Cells lacking nuclei (prokaryotes) vs cells with nuclei (eukaryotes) – Kingdoms (for illustration): Animals Plants Fungi Protista Monera - the prokaryotic organisms  Classification Classification: Kingdom Phylum Class Order Family Used most Genus frequently in Species clinical practise Genus and Species are used whenever we call a bacterium by its name e.g. Staphylococcus (genus) aureus (species) Escherichia (genus) coli (species) , a member of the Enterobacteriaceae (family)  How Bacteria are Classified Characteristics used to classify organisms – Traditional Size, shape, gram reaction, need for O2 Ability to metabolize sugars Metabolic end products – Supplemented by Comparison of 100-300 characteristics Nucleic acid sequence of ribosomal RNA Terms used for Groups of Bacteria by Atmosphere of Growth (in prep…) Aerobic (grows in air), obligate if must have O2. Capnophilic if needs CO2. Facultative anaerobe (grows in air, and can grow without oxygen). Anaerobe (grows without oxygen, and most species do not grow well in air as O2 is toxic for them). Microaerophilic (grows in a low concentration of oxygen, but not in its absence or in air).  Fin. Any questions or comments?  Next lecture! Bacterial growth requirements and metabolism!