Lecture: Bacterial Growth + Sterilisation/ Disinfection

What are key features of bacterial metabolic processes?

• High metabolic rates

• Short generation time

• Ability to compete with eukaryotes when host associated

• Ability to use chemical and physical energy source

How do bacteria replicate?

Binary Fission

Chromosome replicates and cells elongate

Chromosomes separate and cytoplasmic membrane and cell wall invaginates

Cross-wall formation complete

• Not all bacteria form this wall

Daughter cells separate

What are the key features of bacterial growth?

• Increase in number of cells (not the size of cells...)

• Reproduce by binary fission

• Daughter cells of equal size

• Cells may or may not separate after division

• 'String of beads/in chains' - Streptococci

• 'Bunch of grapes' - Staphylococcus

• Produce characteristic morphology

Describe the process of bacterial chromosome replication.

• Chromosome replication initiates cell division

• DNA replicated at origin of replication (oriC)

• Proceeds in both directions, until terminus (ter)

• Which is why bacteria can grow so quickly

• In rapidly growing cells, the newly formed chromosome begins to replicate even before cell division

What are some ways we can measure bacterial growth in liquid media?

In liquid media

• Turbidity

• Absorbance of light = Spectrophotometer

• Light scatter = Nephalometer

• Count particles

• Microscopically: counting chamber

• Electronic particle counter

• Viable counts

What are some ways we can measure bacterial growth in solid media?

In or on solid media

• Serial dilution

How do we get a viable cell count?

• Complete a serial dilution, plating the mixtures and counting the number of colonies per plate at each dilution

• Number of colonies on plate x dilution factor

• = Number of bacteria per ml of culture

What is bacterial generation time?

• The time taken for doubling to occur is called the generation or doubling time

Under optimum conditions, what is notable about the doubling time of bacteria and what is it affected by?

• Under optimum conditions the doubling time is characteristic of each bacteria

• Doubling time is greatly affected by the culture conditions

How does the bacterial growth curve appear?

• Lag phase:

  When bacteria are first introduced to a new environment, they spend time adjusting, synthesizing necessary enzymes, and preparing for rapid division, resulting in minimal cell growth during this period. 

• Exponential (log) phase:

  This is the phase of most rapid cell division, where the bacterial population increases exponentially as cells divide at a constant rate. 

• Stationary phase:

  As nutrients become depleted and waste products accumulate, the rate of cell division slows down and eventually equals the rate of cell death, leading to a stable population size. 

• Death phase:

  Due to the exhaustion of nutrients and the build-up of toxic waste, the number of viable cells begins to decline as cell death surpasses cell division. 

What are some physical, gaseous and nutrient factors that affect requirements for bacterial growth?

Physical Requirements

•Temperature

•pH

•Osmotic Pressure

•Moisture

Gaseous Requirements

•Oxygen

•Carbon Dioxide

Nutrient Requirements

•Carbon

• Nitrogen

•Sulphur

•Phosphorus

•Trace Elements

• Vitamins and Growth Factors.

What are the effects of temperature on bacterial growth?

• Bacteria can grow over a range of temperatures

• All bacteria have an optimum growth temperature

• Most pathogenic bacteria are mesophiles (optimum temp ~ 37°С)

• Temperatures above the optimum growth temp. are more deleterious than lower temps

What are the effects of pH on bacterial growth?

• Bacteria can grow over a range of pH values

• All bacteria have an optimum growth pH

• Most pathogenic bacteria are neutrophiles (optimum pH ~ 7)

• Since pH 7 reflects that of most bodily fluids

• Products of bacterial metabolism are often acidic (lower pH of growth medium)

• Some bacteria grow at pH 2 (stomach acid)

How do we classify bacterial based on pH and their growth?

Alkaliphile

Neutrophile

Acidophile

What are the effects of oxygen on bacterial growth?

• AKA how do we classify their growth based on the amount of oxygen?

Obligate aerobes - Top of tube

• Require oxygen to grow and reproduce

• Use oxygen in cellular respiration to metabolize substances like sugars and fats

• M. tuberculosis + N. meningidis

Obligate anaerobes - Bottom of tube

• Killed by normal atmospheric concentrations of oxygen

• Do not require oxygen for growth

• The presence of oxygen can be toxic to them

• Clostridiodes

Facultative anaerobes - More at top, some at bottom

• Can grow in the presence or absence of oxygen 

• Use aerobic respiration if oxygen is present, but can switch to fermentation if oxygen is absent 

• Staphylococcus + Enterobacteriaeceae

Aerotolerant anaerobes - Grow throughout

• Do not use oxygen for metabolic processes and do not get harmed by it

• Streptococci + Lactobacilli

Microaerophiles - Grow slightly below surface of tube

• Can tolerate only sub-atmospheric levels of oxygen

• Require oxygen but cannot tolerate a high concentration of oxygen

• Campylobacter jejuni

What are some reactive oxygen intermediates (ROI)?

• Superoxide

• Peroxide

• Hydroxyl Radical

What molecules do bacteria produce to detoxify ROI?

• SOD (Superoxide dismutase)

• Catalase

Pathogenic bacteria are ______

• Meaning they require a source of…

Chemoheterotrophs - get their energy from oxidation of organic compounds

• Organic carbon (C)

• Nitrogen (N)

• Sulphur (S)

• Phosphorus (P)

• Potassium (K)

• Magnesium (Mg)

• Calcium (Ca)

• Trace elements

• Iron (Fe), Zinc (Zn), Copper (Cu), Molybdenum (Mo)

• Some require organic growth factors

• NAD or NADP (V factor) some Haemophilus sp and Actinobacillus sp

• (Respiratory pathogens)

What are chemoheterotrophs?

A chemoheterotroph is an organism that obtains its energy and carbon from organic compounds.

What is the Analytical Profile Index?

The Analytical Profile Index (API) is a method for identifying bacteria using biochemical tests. The API system is based on test strips that contain dehydrated reagents and color indicators.

• Provides a numerical code - unique to bacteria

Describe features of bacterial growth and the process, using solid media.

• Agar is added at 1-3% to solidify media

• Media commonly dispensed into petri dishes (plates)

• Bacteria grow as colonies

• Each colony arises from the growth of a single bacterial cell or clump of cells (colony forming unit, CFU)

• Clinical samples inoculated on to agar plates to determine the number of different bacteria present and to isolate single colonies for pure culture and subsequent analvsis

What is enriched media?

Supplemented with nutrients to support the growth of fastidious organisms (bacteria that require specific nutrients).

What are some examples of nutrients added to enrich media?

•Extra Nutrients: Blood, serum, or specific growth factors.

•Promotes the growth of fastidious pathogens (e.g., Neisseria spp., Haemophilus spp.).

What are the different types of hemolysis when using blood agar?

Alpha (a) Hemolysis: Partial breakdown of RBCs, resulting in a greenish or brownish discoloration around colonies (e.g., Streptococcus pneumoniae).

Beta (B) Hemolysis: Complete lysis of RBCs, producing a clear zone around colonies (e.g., Streptococcus pyogenes).

Gamma (v) Hemolysis: No hemolysis or change in the agar (e.g., Enterococcus faecalis).

How is ‘chocolate agar’ produced?

• What is it used for?

(heated blood agar - releases hemin and NAD)

Used for respiratory pathogens.

What is the purpose of selective media?

• What are inhibitors and differential agents?

Inhibit the growth of certain bacteria while allowing others to grow.

• Inhibitors: Chemicals or dyes (e.g., bile salts, antibiotics) to suppress unwanted bacteria.

• Differential Agents: Indicators (e.g., lactose, pH indicators) to distinguish between bacteria based on metabolic activities.

Fungi, like bacteria are __________, they have similar growth to bacteria but may require…

Chemoheterotrophs

• Nutrient-rich media: Fungi require specific media that provide the nutrients necessary for their growth.

• E.g. Common media: Sabouraud Dextrose Agar (SDA), Potato Dextrose Agar (PDA).

Describe the following typical requirements for fungi.

• Temperature

• pH

• Moisture

• Incubation time

• Appearance

•Temperature: 25°C to 30°C

•pH: pH 5.6

•Moisture: Fungi thrive in moist environments.

• Incubation Time: 3-7 days or more. Media are often supplemented with antibiotics (e.g., chloramphenicol) to inhibit bacterial growth while allowing fungal growth.

•Fungi can appear as moulds (filamentous growth with mycelium) or yeasts (unicellular, forming colonies like bacteria).

Describe the steps in the diagnostic process - from sample to diagnosis.

1. Clinical diagnosis + non-microbiological investigations

2. Sample collection

   1. Take specimen correctly, from correct site

   2. Labelling, packaging, transport, storage

3. Staining

4. On plates or in broth

   1. Biochemical testing from pure growth

5. Sensitivities by disc diffusion methods, breakpoints or MICs

   1. DNA / serodiagnosis

What is MALDI-TOF fingerprinting?

Matrix-Assisted Laser Desorption/lonization-Time of Flight

• Mass spectrometry technique

1. Isolated bacteria mixed with matrix

2. Fire laser - giving mass spectrum

3. Compare to data base - allowing bacteria identificaiton

What is sanitizing?

The process of reducing the number of microorganisms on a surface to a safe level

What is disinfecting?

The process of killing or inactivating most or all microorganisms on a surface

What is sterilizing?

The process of killing or inactivating all microorganisms on a surface or object

_____ < _______ < _______

sanitizing, disinfecting, sterilizing

What are physical forms of sterilization?

• Heat

  • Dry (Incineration + Dry Oven)

  • Moist (Pressurized steam + pasteurization; boiling water)

• Radiation

  • Ionizing (.e.g X-rays)

  • Non-ionizing UV

• Filtration

What are chemical forms of sterilization?

• Gas

  • Formaldehyde

  • Ethylene oxide

• Liquid

  • Alcohol

  • Halogens

  • Phenols

  • Aldehydes

Describe how heat sterilizes surfaces.

Causes coagulation of proteins and breakage of Hydrogen bonds

What are the two types of heat used to sterilize?

• How does each work?

1. Dry heat: hot air oven (160C)

2. Moist heat: boiling or autoclaving

   1. Boiling kills vegetative cells, most viruses and fungi within 10 min

   2. Autoclaving

      1. Kills all microorganisms and their endospores in about 15 min

         • 121°C at 15 psi (pounds per square inch)

         • Quicker/more efficient than dry heat

         • Spore killing

Describe how pasteurization sterilizes food/beverages.

Involves heating food or liquid to a specific temperature for a set time and then cooling it rapidly.

• Low-Temperature, Long-Time (LTLT): 63°C for 30 minutes (e.g., traditional milk pasteurisation).

• High-Temperature, Short-Time (HTST): 72°C for 15-20 seconds (common in modern milk processing).

• Ultra-High Temperature (UHT): 135°C for 2-5 seconds (used for milk and dairy products with long shelf life).

What is pasteurization?

A heat treatment process designed to kill pathogenic microorganisms in food and beverages without significantly affecting their quality or nutritional value.

• Developed by: Louis Pasteur in the mid-19th century to prevent spoilage in wine and milk.

**Pasteurisation kills harmful bacteria but does not eliminate all microorganisms.

How does irradiation sterilize?

The major target for these radiations is microbial DNA

What are the two types of irradiation and how do they work?

Ultraviolet (non-ionising) radiation

• Poor penetration power, so microorganisms only on the surface are killed.

• Waves absorbed by many materials, particularly nucleic acids.

• The waves cause the formation of pyrimidine dimers which bring error in DNA replication and cause the death of microbes by mutation.

lonizing Radiation

• X-ray and gamma rays

• lonisation of various substances along with water.

• Results in the formation of many toxic 02 metabolites like hydroxyl radical, superoxide ion, and H20, through ionisation of water.

• These metabolites are highly oxidising agents and kill microorganisms by oxidising various cellular components.

What are the common liquid chemical disinfectants and how does each function?

Sodium Hypochlorite (Bleach):

• A widely used disinfectant for surfaces in hospitals, homes, and laboratories.

• Effective against a broad range of bacteria, including Staphylococcus aureus, E. coli, and Salmonella.

Hydrogen Peroxide (H2°2):

• Used for surface disinfection, medical device sterilisation, and wound care.

• Kills bacteria by producing free radicals that damage proteins and cell membranes.

Alcohols (Ethanol, Isopropanol):

• Used for skin disinfection and cleaning surfaces in healthcare settings.

• Effective against many bacteria, particularly Gram-negative and Gram- positive species, by denaturing proteins and disrupting cell membranes.

Quaternary Ammonium Compounds (Quats):

• Used for surface disinfection in hospitals, food service environments, and household products.

• Effective against a variety of bacteria, but less effective on spores and some racictant atroine

What are the common gaseous disinfectants and how does each function?

Ethylene Oxide (EtO):

1. Commonly used for sterilising medical devices, surgical instruments, and sensitive equipment.

2. Effective against a wide range of bacteria, including spores.

3. Used in low temperatures, making it suitable for heat-sensitive materials.

Ozone (03):

1. A strong oxidiser used to disinfect air, water, and surfaces.

2. Effective in killing bacteria by disrupting their cell walls and internal structures.

3. Commonly used in water treatment and some hospital settings.

Chlorine Dioxide (ClO,):

1. Used as a disinfectant for air and surfaces in healthcare facilities and food processing plants.

2. Effective against bacterial spores and vegetative bacteria.

What materials require sterilization?

• Microbiological and tissue culture.

• Medicines.

• Surgical, optical and electrical instruments.

• Reagents.

• Equipment or containers in which any of the above are to be handled.

• Infected material.

What factors may affect the action of disinfection?

• Period of exposure

• Concentration

• Temperature

• pH

• Organic matter

• Number of bacteria present