microbio

micro bio

microbes - all microorganisms (algae etc, not just bacteria)

microbial disease

  • destroy living cells/tissues of infected organisms

  • release chemicals that interfere with normal activities of cells

uses of microbiology

  • bioterrorism

    • refers to biological agents 9microbes or toxins) used as weapons to further personal or political agendas

  • therapeutic agents

    • Health products

    • Vaccines

    • Drugs

    • gene therapy using virus

  • factories → insulin (bring glucose level down)

  • sewage treatment → bacteria used to break down oil into smaller pieces(Bio-remediation)

Summary

negative

  • infectious agents

  • food spoilage

positive

  • therapeutic agents

  • used in the manufacturing of industrial

  • food products

  • bio-remediation, e.g. sewage treatment

Different shapes of microorganisms


Identification of microorganism

  • group/family

  • genus

  • species

  • strain

    • a subset of a bacterial species with slight differences from the other bacteria of the same species → Mutation of viruses

naming system

  • species names are usually derivatives of some descriptive properties

  • genus name first, species name last

Staphylococcus aureus

When typewritten: italicized when handwritten: underline

Gram staining

  • the ability of the cell wall to retain the crystal violet solvent based on thickness of cell wall

  • iodine prevent crystal violet from being washed off

  • Gram-positive

    • Purple → Absorbed by the higher levels of peptidoglycan in the cell wall

    • 90% peptidoglycan

  • Gram-negative

    • Red → Absorbed by the lower levels of the peptidoglycan in the cell wall

    • 10% peptidoglycan

    • got the layer ontop

Bacterial isolation

16-streak method

  • To get a pure culture

  • it is impossible to study the characteristics of each species of bacteria unless they are isolated and maintained as pure culture

  • help to isolate one bacteria species

Significance of “pure colonies”

  • Each colony originate from one single bacterial cell

  • pure colony: colony that contains one STRAIN of microorganism

  • any study requires pure strains as a start

  • Each colony originates from one single bacterium which divide and reproduce asexually via binary function

  • pure means only one type, no others

  • Understand the morphology of the bacteria

Tools

  • inoculating loop → heat until glowing red hot

Steps

  1. flame the inoculating loop until red-hot

    • remember to heat it each time using, and wait for it to cool down (touch on agar plate)

  2. dip the inoculating loop in a medium/source containing mixed culture

  3. streak bacteria across the agar plate in the following manner → flame your loop in between each series

  • Example of 16-streak on agar plate


How to confirm that this colony is pure?

  • perform another round of 16-streak with that colony

Important concept

  • 1 colony = 1 single bacterium at time of plating CFU = no. of bacteria that can form a colony

Counting bacteria

  • concentration of bacteria in the original sample(before dilution)

  • colony forming unit: a unit to indicate the number of bacteria that is capable of multiplying into a colony

  • continue to dilute the sample so that the number of colonies on spread plate is < 300

Quantifying bacteria

1ml = 1000 micro litres

Aseptic technique

  • a set of specific practices and procedures performed under clinical and lab settings to prevent the contamination of unwanted microorganisms

  • Complete all operations as quickly as possible

  • All materials must be sterile

  • Disinfect work area

  • Pipette tips can not come into contact with any surfaces

  • vessels must be open for the minimum amount of time possible

  • on opening a test tube or bottle, the neck must be immediately warmed by flaming

  • While vessels are open, all work must be done close to a bunsen burner flame where air currents are drawn upwards → convection current

  • Windows and doors must be closed

  • Do not talk (or breathe >< !!)

  • All items that come in contact with the bacterium must be sterilised

Pouring of agar plate

  • neck of agar bottle is passed through the flame

  • petri dish lid is opened as little as possible, angled and kept over the base

  • sterilised molten agar is poured in and left to set

  • each petri dish holds ab 20ml, so 200ml fr 10 plates

  • solidified agar is brought to laminar flow hood to air dry


Bacterial growth and the culturing of microbes

  • Binary fission

    • Cell enlarges, and the DNA replicates

Growth curve

Lag phase

  • Adapting to the new environment

  • build-up of growth “tools”

    • e.g. enzymes

  • cells increase in size but do not divide

  • 4 parts

    • lag phase → bacteria adapting to the environment so v slow

    • log exponential phase → highest reproduction and metabolic rate because they “settle” in

    • stationary phase → slow down in growth due to depletion of nutrients and increased metabolic waste products

    • death phase →Exponetial cell death

Bacterial enumeration

Total cell count

  • Uses a Haemocytometer

  • Volume of liquid in counting grid

  • At least 10 million bacteria for accuracy

Viable cell count

  • methods

    • plate count

    • membrane filtration

    • most probable number (MPN)

  • 16 streak technique

  • 1 colony = 1 single bacterium at time of plating

  • cfu = no. of bacteria capable of forming a colony

  • 30 - 300 count plate

Macronutrients

  • Large amounts

Micronutrients

  • Trace elements(Small amounts)

Culture media:

Define media

  • all components and the concentrations are known

Complex media

  • contain some ingredients of unknown composition and/or concentrations

Media elements

  • Peptones

    • water-soluble proteins

  • extracts

    • aqueous extracts → using made of beef or yeast to supply nucleic acids, fats, polysaccharides as well as vitamins and trace minerals

  • nutrient broth vs nutrient agar

    • one as agar one dh → turn medium semi-solid

    • both need to be autoclaved before used

    • broth → cultivating large amount of clear colonies

    • agar → counting colonies

Motility test

  • see how fast bacteria can grow

Classification of bacteria

  • morphology

  • staining (based on cell wall)

  • environment factor

  • nutrient requirements

  • biochemical tests

Purpose

  • to be able to culture/control microbes to identify microbes correctly

colony morphologies

  • colour

  • size, elevation, texture and margin

  • surfaces

KOH test - easier than gram staining

  • rapid test to determine gram reaction

  • gram-negative

  • thinner cell wall

  • easily lysed by 3% [potassium hydroxide

  • DNA strands exude from cell

  • visible lifting → got the strands

Antibiotics

  • Kill bacteria

  • No effect on virus → only bacteria

    • in order to defeat virus, need to kill the host

  • selectively toxic

    • either kill them (bacteriostatic)

    • inhibiting their growth (bacteriostatic)

    • no harm to patient

  • Broad spectrum vs Narrow spectrum

    • Broad covers most of the bacterial attacks of an illness

antibiotic side effects

  • certain antibiotics w side effects when taken by patients

  • side effects.toxicity occurs rekrenfdsjkh

jason tan skipped fill in

ase → enxymes

Transpeptidase - > links the peptidoglycan layers

antibiotic tzarted DNA at lag phase

DNA synthesis → RNA synthesis

Protein synthesis

  • DNA cannot come out → Synthesises to get RNA

nucleic acid ref to DNA and RNA

transcription → nucleus translate → cytoplasm

tetracycline binds 2 ribosome → antibiotic

prevent peptide bond from forming btwn amino acids

Quinolones(Bactericidal)

30S Binding to the tRNA o inhibit protein synthesis

DNA replicates by unwinding its Helix into RNA

DNA cannot unwind

only gram-negative gt that layer

Polymyxins → Bind to LPS and phospholipids

enivnronmentl factors

  • pH

  • temp

  • o2 requirements

pH range

  • Neutrophiles

    • Growth optimum between

  • acidophiles

  • growth optimum between ph 0

Oxygen requirements(Catalase test):

Obligate aerobe

Facultative anaerobe →Felexible: even when there is no oxygen, they can still live

Strict anaerobe → Cannot grow when there is no oxygen

Aerotolerant anaerobe

break down hydrogen and water → da bubble more like da baby

classification of microorganisms based on carbon and energy sources

  • nutrient requirments

  • antibacterial test

Due to dff species in genetic makeup, microbes ha ve different capabilitys/preferences in obtaining g energy from various nutrient sources

Autotroph

  • make their own food

  • Photoautograph

    • an organism that uses sunlight as an energy source and C02 as a carbon source

  • Chemoautotroph

    • oxidises inorganic compounds as an energy source and CO2 as their carbon source

    • Synthesise their own organic molecules

catalase

  • bubble → aerobic respiration

  • no bubble → does not undergo aerobic respiration

starch hydrolysis test

  • Detect presence of amylase and glucosides

  • detects if bacteria absorbs the starch: blue black

Urease test

  • Detect….

  • Yellow→ Negative result(acidic)

  • Orange→ Uninoculated bacteria

Metabolic waste

  • Gas formation(CO2, Methane)

  • Salmonella

Heterotroph

  • cannot make their own food, feed on other organisms

  • photohetertroph

    • energy source: sunlight

    • carbon source: organic compounds from the environment

  • chemoheterotroph

    • energy source: oxidises organic/inorganic compound

    • carbon source: organic compounds from the environment

      • chemolithoheterothrophs

catalase

  • bubble → aerobic respiration

  • no bubble → does not undergo aerobic respiration

starch hydrolysis test

  • Detect presence of amylase and glucosides

  • detects if bacteria absorbs the starch: blue black

Urease test

  • Detect….

  • Yellow→ Negative result(acidic)

  • Orange→ Uninoculated bacteria

Rapid test kits:

  • Miniaturised “test tubes”

  • Capsules contain dried chemicals

  • Multiple enzymes tested simultaneously