1st test from plennary Flashcards
What are the 2 methods when detecting bacteria in a sample
Direct and Indirect way
What are the Aims of culturing bacteria
• Isolation of causative agents from pathological samples
(DIAGNOSTIC AIM)
• antibiotic susceptibility testing
(for the purpose of targeted ANTIBACTERIAL THERAPY!!!)
• RESEARCH purposes
• INDUSTRY
(food industry, pharmaceutical industry, fermentation, antibiotic production etc.)
What do you do referring to the DIAGNOSTIC AIM
Isolation of causative agents from pathological samples
What is the purpose of targeted antibacterial therapy
antibiotic susceptibility testing
When performing Detection of bacteria in a sample the Direct way - What are the different types?
- Smear
- Impression Smear
- PCR
From where can you collect samples for a SMEAR when performing the direct way of bacterial sample
- Nasal discharge,
- Vaginal discharge
- Pus from an abscess,
- Mastitic milk,
- Blood (septicemia),
- Urine
What is impression smear? Example of a examination with an impression smear
Quick and simple (shape, size, staining, mixed infection
(eg. anthrax: parenchymal organs – cutted surface
What is the advantage of an impression smear
advantage: quick and simple,
What is the disadvantage of an impression smear
disadvantage: only: quantity, shape, size of bacteria
What is PCR and what do we use it for
PCR (detection of specific DNA-sequences)
– unculturable bacteria!
What is this

agar-gel electrophoresis

Smear
(sputum sample)

Smear, wound secretion
(anthrax, Bacillus anthracis)

Smear (cattle, spleen),
toluidine-blue staining
(Bacillus anthracis)

Smear from a deep anaerobic wound
(Clostridium tetani)

Smear, sheep gastrointestinal tract,
Gram-staining
(Clostridium perfringens)

Smear, mastitic milk
(Streptococcus agalactiae)

Smear, mastitic milk
(Streptococcus sp.)

Smear, human genital secretion
(Neisseria gonorrhoea)

Smear, human vaginal secretion
(Lactobacillus sp.)

Smear, urinary sediment, dog
(E. coli)

Smear, human blood
(plague, Yersinia pestis)
How do you perform detection of bacteria in the
Indirect way
- By animal trial
after death or extermination, we try to isolate the causative agent,
**- Inoculation the sample onto a medium** we culture (isolate) bacteria on a medium, preparing pure culture, identification.
How can we culture Treponema pallidum?
INDIRECT METHODE
- In rabit testicle ONLY
- Treponema pallidum: human syphilis: culturable in a rabbit testicle „only”.
How can we culture Francisella tularensis
INDIRECT WAY
Francisella tularensis: tularemia, inoculation into a MOUSE
Composition of media
Bacteria does not need to be in harmony with the media
TRUE or FALSE
FALSE
The connection between bacteria and media is very closed. Must be in harmony with demand of bacteria!
What is the composition of Media
Water
C-sources
N-sources:
- vitamins and other additives
Osmotic pressure
pH: 7.2-7.4
80-90% of the bacterial cell is water!
True or false
True
Which organic C-source is needed
GLUCOSE!!! (Pyruvic acid!)
Composition of media: N-source
N-sources:
- protein-hydrolisates: peptone (digested casein), triptone (digested muscle)
- native protein: several species need it: eg. Trueperella pyogenes
Composition of media- vitamins and other additives:
B1 vitamin has a impact on which production
- vitamins and other additives:
- *- B1 vitamin:** bacteria which have impact in cheese production
Composition of the media, vitamins and aditives
- B2 vitamin has an impact on:
most Lactobacilli
- V (NAD, X-factor (haemin has a impact on which species?
- V (NAD, X-factor (haemin: Glaesserella, Avibacterium, Actinobacillus spp.
- Brucella: Which components are needed
- Brucella: B1 vitamin, nicotinic-acid-amide, pantothenic-acid is needed
- Erysipelothrix rhusiopathiae: Which components are needed
- Erysipelothrix rhusiopathiae: para-amino-benzoic-acid (PABA) is needed
Composition of media
OSMOTIC PRESSURE
In a par with 0.85% NaCl solution
COmposition of media
PH
- pH: 7.2-7.4
Classification (grouping) of media
According to
- ORIGIN
- STATE
- AIM of CULTURE
Classification (grouping) of media
According to • Origin
1- natural (potato slide, blood, serum, milk, bile, urine)
2- artificial
3- synthetic (=chemically defined) - the exact amount of ingredients are known
Classification (grouping) of media
According to
State
(liquid, semisolid, solid)
Classification (grouping) of media
According to
Aim of culture
1- common (=basic nutrient) - these are capable of sustaining growth of the less fastidious bacteria
2- selective - contain inhibitory substances that prevent the growth of unwanted bacterial species
3- differential (=indicator) - They are designed to give a presumptive identification of bacterial colonies due to the biochemical reactions in the media. They often contain a fermentable sugar plus a pH indicator that gives a colour change in the media.
Bacterial growt show what in a liquid samlpe
TURBIDITY
Sterile are clear
Which bacteria

Mycobacterium phlei
Name arrows from top to bottom

- Pellicle
- No turbidity
- Sediment
Which bacteria

Staphylococcus aureus
Name bacteria

Bacillus subtilis
Name bacteria

Escherichia coli
Which medium is made from Sea weed
Agar - Agar
Which bacteria can be cultured in agar agar
Gelidium sp., Gracilaria sp.
Name a special characteristic of the agar agar powder mediun
DIFFERENT MELTINGPOINT and FREEZING Point
Agar-agar powder
DIFFERENT MELTINGPOINT and FREEZING Point
in agar agar powder, but what is is
melting-point:
85-90 °C
solidifying-point:
45-50 °C
Semi solid test are used for
Motillity test
Which media
Which test
Which is positive and negative

SEMI SOLID MEDIUM
MOTILLITY TEST
LEFT is NEGATIVE and RIGHT is POSITIVE
Name of test in which medium

Oxidation/Fermentation test
– semisolid medium!
(Do NOT mix with oxidase test!!!)
Left medium

SOLID MEDIUM
Blood agar
10% sheep or ox blood
Solid, artificial, differential medium
Solid Medium
Right medium

Solid, Artificial, Common medium
Nutrient agar

Non-haemolytic colonies (Pasteurella multocida)

Haemolysis
Streptococcus pneumoniae
alpha (partial, incomplete, biliverdin)

Hemolysis
Streptococcus equi
Beta (compleete)

Carbohydrate broths
Mannitol, Dextrose, Lactose, Sucrose
Liquid, artificial, differential medium
(Phenol red – pH indicator)
UNINOCULATED - NEGATIVE - POSITIVE
Inhibitory materials:
pH indicator:

MAC CONKEYS AGAR
Solid Artificial Selective Differential medium
Lactose neg - inoculated - positive
Inhibitory materials:
- bile salts crystal violet
pH indicator:
- neutral red




PREPARATION of MEDIA
Dehydrated media
- wide range
- easy to use
- dissolved in bidest. water, boiled - Sterilisation (autoclaving!)

AUTOCLAVE!!! (121degreed, 15 minutes)

Plastic Petri-dishes
- Gamma-steril (irradiated)
- 90 mm in diameter
- 20 ml medium

BLOOD AGAR
10% defibrinated sheep or ox blood
Add 10% blood
to the cooled medium (45-50 °

Chocolate agar
Heat treatment:
8 0 ° C , in 20 m i n
Bacterias suitable for chocolate agar
Avibacterium paragallinarum
Actinobacillus pleuropneumoniae
Glaesserella (Haemophilus) parasuis
Taylorella equigenitalis
Histophilus somni

Inoculating loop!!!

Inocculating loop and bunsen burner

Fowl typhoid
(Salmonella enterica
(st. Gallinarum))

Inoculation of a sample onto the surface of a solid medium
- *- specimen** (liver, spleen, lung, pus, mastitic milk, urine)
- *- Petri dish** is first labelled, on the agar side
(type of specimen, date of inoculation - waterproof marker pen)
- burn the surface of the organ with a hot plate shaped metal instrument (e.g. with an old scalpel), decontamination
- stab with the inoculating loop in the organ through the burned surface
- pull out the inoculating loop from the organ, and streak the agar plates

Laminar flow box (biosafety cabinet)

Incubator (37 °C)
What kind of culture

PURE CULTURE!!!
- descendants of the same bacterium cell
- same genotype
- same phenotype (cultural, morphological, biochemical characteristics)
What heppend here

Airborn contamination
What are the circumstances of incubation
- atmosphere
- temperature
- time of incubation
What does Obligate aerobe bacteria mean
They can grow in the presence of O2 only.
Name Obligate GRAM + bacterias
- Bacillus
- Dermatophilus • Micrococcus
- Nocardia
- Rhodococcus
Name Obligate GRAM - bacterias
- Brucella
- Bordetella bronchiseptica • Francisella
- Moraxella
- Pseudomonas
Staining of Obligate bacterias
MYCOBACTERIUM
ZIEHL- NEELSEN staing
What does obligate anaerobe bacteria mean
They can not tolerate O2 at all.
Name Obligate anaerobe Gram + bacteria
- Actinomycesbovis
- Peptostreptococcus
• Clostridium
Name obligate gram - anaerobe bacterias
- Bacteroides
- Dichelobacter
- Eubacterium
- Fusobacterium
- Brachyspira
- Campylobactermucosalis
What does
Aerobe, facultative anaerobe bacteria mean
They can grow in the presence or in the abscense of O2, too.
What are the facultative anaerobe GRAM + bacteria
Corynebacterium
Erysipelothrix
rhusiopathiae
Listeria
Staphylococcus
Streptococcus
What are the facultative anaerobe GRAM - bacteria
• Actinobacillus
• Aeromonas
• Enterobacterium
• Haemophilus
• Pasteurella
• Vibrio
What is
Microaerophile bacteria
4-6% partial O2 pressure is needed
Microaerophile GRAM + and GRAM -bacteria
GRAM +
- several Actinomycessp.
- Lactobacillus
GRAM -
• Campylobacter spp.
What is
Capnophile bacteria
They need 5-10% CO2
Caphnophile GRAM - bacterias

Caphnopile GRAM + bacterias
Gram+
- Actinomycesviscosus
- Dermatophilus congolensis
What does the picture show

Where the bacteria grow
- AEROBE
- Microaerophile
- Faculative Anaerobe
- Anaerobe
Anaerobic cultures
Anaerobic culture
- candle jar method
- chemical
- pirogallic acid + KOH
- H2 + palladium catalisator
- Na-borohidrid + NaHCO3 + ascorbic acid + H2O •other
•evacuation of air with a vacuum pump
- anaerobic broth
- deep semisolid agar
- pre-reduced media

Light microsope
visible light (400-700 nm) immersion objective and imm. oil resolving power: 0.2-0.4 μm magnification: 1200-1500×
How is this image taken

Darkfield microscope
Special condenser, illuminate with oblique ray of light, dark background, corpuscular elements (bacteria) are glittering,
examination of bacterial motility
What do we examine with dark field microscope
examination of bacterial motility