Microbial cultivation Flashcards
Cultivation techniques and appropriate media
It is the process of growing microorganism in culture by taking bacteria from the infection site (in vivo) and growing them in an artificial environment (in vitro)
Microbial Cultivation
Purposes of bacterial cultivation
- To grow and isolate all bacteria present in clinical specimen
- To determine which of the bacteria that grow are most like causing infection and which are likely contaminants or colonizers
- To obtain sufficient growth of clinically relevant bacteria to allow identification and characterization.
this is a mixture of nutrients needed by bacteria to grow in vitro
Culture medium
A culture may be:
a. pure culture if only one type of organism is present or
b. mixed culture if populations of different organisms are present
Phases of culture media: According to Consistency
Solid form - agar plate; with the base agarose
Liquid form - broth
Semisolid form - w/ 0.5% or less agarose content
Biphasic- solid and liquid phase may be used.
Survival of bacteria in a culture medium depends on:
Availability of essential nutrients and appropriate environmental conditions
A bacteria that needs relatively complex requirements and exceptional media components to aid its growth
A fastidious organism
is defined as any organism which has very complicated nutritional requirements, meaning it will not grow without specific factors present or in specific conditions.
bacteria that do not need special nutritional supplements and conditions to grow on agar plates.
Nonfastidious bacteria
What happens to bacteria in broth growth media
- nutrients are dissolved in water, and bacterial growth is indicated by a change in the broth’s appearance from CLEAR to TURBID
What do more growth iof bacteria in broth culture media indicate
-indicates higher cell density and greater turbidity
Thioglycollate broth is an example of what type of culture medium
Semisolid medium
- thiglycollate broth (liquid) also contains small amount of agar
Thioglycollate broth is an example of what type of culture medium
Semisolid medium
- thiglycollate broth (liquid) also contains small amount of agar
Other than amount of growth present in culture media, ________ of growth within a broth provides an indication of the type of organism present based on oxygen requirements.
- location of growth
e. g. strict anaerobes will grow at the bottom of broth tube whereas aerobes will grow near the surface.
microaerophilic organisms will grow slightly below the surface where oxygen concentration are lower than atmospheric conc.
facultative anaerobes and aerotolerant organisms will grow throughout the medium since they are unaffected by variation in oxygen content.
A medium in which a solidifying agent is added to nutrient and water
Solid medium
The most common solidifying agent
Agarose
-has a unique property of melting at high temp (>95 °C)
and re-solidify after temp falls below 50°C
The petri dishes containing the agar is referred to as
-Agar plate
Types of culture
Pure culture- comes from one type of microbe/ the same species
Mixed culture- more than one type of microbe/ more than two species maybe across kingdoms
Stock culture - unwanted microbe is present; contaminants are fungi and other bacteria
……..
Examples of Solid media - contains 2-3% agar
Triple sugar iron agar (TSI)
Blood agar plate (BAP)
Chocolate Agar plate (CAP)
Mckonkey agar plate (MAC)
Agar is a sulfated polymer made up of________________
Carbohydrates
D-galactose, 3,6-anhydro-L galactose, and D-glucuronic acid derived from red algae
Example of Liquid Medium - does not contain agar
BHI - Brain-Heart- infusion medium
TSB- Trypticate Soy broth
Thioglycolate broth
Example of Semi-Solid medium - agar content is only 0.5-1%
SIM - Sulfide indole motility media
Type of medium necessary for gas testing. When you want to know certain bacteria that produces gas
Semi-solid medium
-SIM
Three test that can be observed when using SIM or sulfide-indole motility medium which is a semi-solid medium
- Black precipitate (due to sulfide)
- Indole production (gas)
- Motility (growth outside line of inoculation)
What is the indication of bacterial growth in liquid medium
e.g. in TSB, BHI and thioglycolate
-Changes in transparency of liquid such as turbidity and cloudiness
Melting point of agar _________
Solidifying temp ___________
Cooling temp ___________
- 80-90 °C
- 40-50 °C
- 55-60 °C
Types of culture medium according to COMPOSITION
Synthetic or defined medium
Non-synthetic or complex medium
Tissue Culture medium
A type of culture medium according to composition in which all the components are KNOWN
Synthetic or defined medium
Types of culture medium according to composition in which some substances in the medium are UNKNOWN and are also very useful for isolation of medically significant bacteria
Non-synthetic or complex medium
e.g. TSB, NB (nutrient broth), and MAC
A type of culture medium according to composition used for obligate intracelluar bacteria or hrd to grow bacteria such as tryponema, rickettsia, and chlamydia
Tissue culture medium
The steps required prior to examining bacteria in/on culture media are:
Sterilization of culture medium
Selection of culture medium
Inoculation of culture medium
Incubation of culture medium
Ways of inoculation method in vivo
- Chorioallantoic membrane inoculation
- Amniotic inoculation
- Yolk sac inoculation
- Allantoic inoculation
Type of Culture medium according to the dispensing or distribution method for the medium
Plated media- petri dish
Tube media
Variations of Tubed media
Broth liquid - liquid all throughout
butt - there is no slant (murag tunga)
Agar-slant - naa syay murag half
Special slant- mix of slant and butt
Type of Culture medium according Use
Simple Media/ General purpose media/ Supportive media Enrichment Media (Liquid) Enriched Media (Non-selective media) Differential Media Selective Media
Examples of Simple media: can grow any non-fastidious bacteria
Nutrient Agar (NA) Nutrient broth (NB) Trypticase Soy broth (TSB)
A type of culture media according to use in which any non-fastidious bacteria can grow and there is no additional supplements added in the media
Simple/General/ Supportive media
A type of culture media according to USE which contains SPECIFIC nutrients required for the growth of specific bacterial pathogens that may be present alone or with other bacterial species in the specimen.
- for fastidious organism
Enrichment media
-specific nutrients without additional supplements (Liquid)
Enrichment Non-selective with additional supplements such as blood, vitamins, and yeast extracts (Solid)
Considered as an enrichment selective media that contains antibiotic for gram-negative bacteria– meaning that it is only used for the growth of gram-positive cocci
LIM broth/ Remel Todd Hewitt broth
Considered as an enrichment selective media used for the cultivation of gram-negative enteric organisms.
Gram-negative broth (GN broth)
Kind of blood used in Blood agar plate (BAP)
Non-hemolyzed blood
Kind of blood used in Chocolate Agar plate (CAP)
Hemolyzed blood - that’s why the brown color
BAP (Blood agar plate) is used to
-detect hemolytic patterns of streptococci
can be simple and enriched or differential medium
MAC (Mckonkey agar plate) is used to detect
-fermentation of sugar
Lactose fermenters - Pink - UTI causing bacteria
Non lactose feementers- Colorless - GIT infection
pH indicator for MAC (Mckonkey agar plate)
Neutral red
Hemolytic pattern also called as “complete hemolysis”, it incorporates a complete lysis of red cells in the media around and under colonies.
Beta-hemolysis
Hemolytic pattern also called as “partial hemolysis”. It appears green due to the hydrogen peroxide produce by the bacterium, oxidizing hemoglobin to methemoglobin
Alpha-hemolysis
Hemolytic pattern which is characterized by the “absence of hemolysis” or no hemolysis at all in the area around a bacterial colony. This appears brownish
Gamma-hemolysis
Examples of Lactose Fermenting bacteria
Escherichia coli and Enterobacter aerogenes
Examoles of Non-lactose fermenting bacteria
Proteus vulgaris, Salmonella typhimurium, Staphylococcus aureus
Type of culture media according to use in which the media is incorporated with antibiotics, dyes, chemicals, to inhibit growth of microorganism while promoting the growth of desired organism.
Selective Media
The two products of fermentation
-Alcohol and CO2 gas
Change of color in culture media from red to yellow during fermentation is caused by
-Production of CO2 - dissolving it in H2O- formation of Carbonic Acid - change red to yellow
Change of color in culture media from red to yellow during frmentation in a Manitol Salt Agar (MSA) is caused by
Note: Only S.aureus is Manitol fermenting
-Production of CO2 - dissolving it in H2O- formation of Carbonic Acid - change red to yellow
Thiosulfate citrate-bile-salts-sucrose sugar (TCBS) is a special media used for culture of what bacteria?
Vibrio spp.
- media must be boil only, never autoclave
Term for the introduction of the sample/specimen into the culture medium
Inoculation
The three inoculation techniques
Streaking
Stabbing
Overlapping
The most common inoculation technique and is done by gently rolling the tip of the swab onto the upper portion of the plate
Streaking
An inoculation technique used for antimicrobial susceptibility testing and also called as “disk diffusion method”
Overlapping inoculation method
In streak plate method, what is the most concentrated area on the plate
The 1st quadrant
-but still depending on the density of the bacteria
In streak plate method, what is the most diluted area in the plate?
The 4th quadrant
-but still depending on the density of the bacteria
Stab and stroke culture is for what type of culture media according to consistency
Liquid media- Special slant (w/ butt and slant portion)
