WEEK 4

BMY3101-1 MICROBIOLOGY I

WHAT HAVE I LEARN?

This week, we continue on the electron microscope. Dr ask a question about what to do and how to treat the superbug which are resistant to antibiotic. We form groups to discuss about this question. A few representative of group gave some interesting method for example which involve genetic engineering, and also some relate to the "lock and key" hypothesis. The answer is " find another type of antibiotic". We then study about preparation of specimens for light microscope.

We divide into groups to discuss among ourselves for the classification of organisms topic. After that we exchange our knowledge with other friends and teach them what have we discovered. Actually i am quite confused when listening to friend's ideas, this method can really make us to be proactive but then i don't really understand all the explanation, and maybe the examples given are wrong but we don't know. I hope at least Dr can give more details on this topic.

Electron Microscope

- employs a beam of electron in place of light wave to produce the magnified image.
- resolving power 100 times than light microscope

Scanning electron microscope ( SEM)

• Produce a 3D image 
• magnification 1000X to 10 000X
• use to study surface features of cells and viruses
• phagocytosis process
• treatment ( anitbiotic ) - lyse the cell wall of virus
  compare shape of bacteria before ( not treated, normal cell) and after
  *make sure the same magnification is used

Transmission electron microscope ( TEM)

• image produce are 2D
• can see cross section
• magnification - 10 000X to 100 000X

Electron cryotomography

• rapid freezing technique preserve native state of structures examined in vacuum
• 3D structures
• provide extremely high resolution images
• uses liquid nitrogen ( -175°C to -195°C)

Magnetosomes bacteria

• orient along the magnetic field lines of Earth's magnetic field.
• have organelles called magnetosomes that contain magnetic crystals.
• ferum crystal aline in body ( bacterial cell )
• found in the sea

Preparation of specimens for light microscopy 

• Wet mount
  demonstrating motility or structure of microorganisms
• Smears

1. Preparing staining
   Thin suspension of cells placed on glass
   no cover slip needed
   air dry completely
2. Fixation
   Preserves internal and external structures and fixed them in position
   - heat fixing
   - chemical fixing
3. Staining 

• Simple staining
• Differential staining - detect presence or absence of structures
• special staining

Dyes or stains

• chromophore - colourful
• to increase contrast with background, make cell more visible

Simple staining

• reveals cell shapes and arrangements
• acidic dyes
  eosin & picric acid
• basic dyes
  crystal violet, methylene blue, malachite green and safranin

Differential staining

Gram Staining 

• divide based on cell wall structure
• gram positive and gram negative
• must use single colony only can classify them
• before apply safranin the cell will be in transparent
• counterstain is to differentiate whether gram positive or gram negative

Why gram negative is more harmful?

Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer which hides the antigens of the cell and so acts as "camouflage". Ability to produce endotoxins ( beta- lactamase inhibitor) which is antibiotic resistance genes .

Lipopolysaccharides (LPS), also known as lipoglycans and endotoxins, are large molecules consisting of a lipid and a polysaccharidecomposed of O-antigen, outer core and inner core joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria.

Beta- lactamase inhibitor

Beta-lactamases are a family of enzymes involved in bacterial resistance to beta-lactam antibiotics. They act by breaking the beta-lactam ring that allows penicillin-like antibiotics to work. ( enzyme that degrate antibiotic) .Beta-lactamases (β-lactamases, also known as penicillinase) are enzymes (EC 3.5.2.6) produced by bacteria, that provide multi-resistance to β-lactam antibiotics such as penicillins, Cephalosporins, cephamycins, and carbapenems (ertapenem), although carbapenems are relatively resistant to beta-lactamase. Beta-lactamase provides antibiotic resistance by breaking the antibiotics' structure. These antibiotics all have a common element in their molecular structure: a four-atom ring known as a β-lactam. Through hydrolysis, the lactamase enzyme breaks the β-lactam ring open, deactivating the molecule's antibacterial properties. Beta-lactamases produced by Gram-negative organisms are usually secreted, especially when antibiotics are present in the environment.

Pathogenicity

Pathogenic - virulent ( extremely severe or harmful in its effects )

Actinomyces is a genus of the Actinobacteria class of bacteria. They are all gram-positive. They are known for the important role they play in soil ecology. The smell after rain is produce by this bacteria.

Acid- fast staining 

( Ziehl - Neelson Stain)

• use for fassy microbes ( which difficult to cultured in the lab)
• useful for genus Mycobacterium 

Special staining 

Negative staining 

• colourless bacteria against colored background 
• visualize capsules surrounding bacteria
• capsule repels stain
• capsules are colourless against a stained background

Endospore staining 

• use for clostridium and bacillus 

Flagella staining

• to enhance the flagella

Classification of Organisms 

Taxonomy 

• taxis - arrangement or order
• nonos or nemein - law
• govern - to distribute

Defining and naming groups of biological organisms on the basis of shared characteristics. ( mutual similarities) 

Classification 

• arrangement of organisms into groups based on mutual similarity or evolutionary relatedness

Phylogenetic - evolutionary development of organisms

Phyletic classification - evolution ( slight changes ) < more specific 

Direct comparison of genetic materials and gene products( hair black or white )

What makes phylogenetic classification possible

1. Highly conserved genetic sequence ( very small evolution )
2. Advancement in sequencing technique ( about the PCR to know DNA sequencing

Phylogenetic tree:

Evolutionary relationships among group of species

Tree constructed based on molecular attribute ( traits )

eg: wings ( chicken, bird )

Numerical taxonomy 

• grouping by numerical methods of taxonomic units based on their character states.
• creates a taxonomy using numeric algorithms like cluster analysis rather than using subjective evaluation of their properties