Microbiology Lab Practical Midterm

Bright field Microscope

A type of microscope that allows light rays to pass directly to the eye and is most commonly used by students

Ocular

This part of a bright field microscope is known to be the eyepiece that consists of two or more internal lenses with a usual magnification of 10x; binocular

Objective Lens

This part of the bright field microscope has magnifications of 4x(rapid scan), 10x(low power), 40x(high-dry), and 100x(oil immersion)

Nosepiece

This part of the bright field microscope allows for the objective lens to be rotated into position over the slide

Total Magnification

This process is determined by multiplying the power of the objective lens times the power of the ocular lens used

Condenser

This part of the bright field microscope is located under the stage which collects and directs the amount of light from the light source to the slide being studied

Diaphragm Control

This part of the bright field microscope is located within the condenser which regulates the amount of light that reaches the slide; can either be controlled by turning a knob or a lever

Course Adjustment Knob

This part of the bright field microscope is found on the side of the microscope which moves the slide up and down considerably for better focus; does not get used again after 10x objective lens

Fine Course Adjustment

This part of the bright field microscope is found on the side of the microscope which moves the slide up and down minimally for finer detail

Resolving Power

The ability of a lens to to completely separate two images in a microscopic field

Steps for Oil Immersion

1.) Make sure slide is in focus under microscope

2.) Turn the nose piece halfway between the 40x and 100x objective lens

3.) Apply a drop of oil to the slide

4.) Fully rotate the nosepiece until the 100x objective lens rests on top of the slide

5.) Adjust the fine adjustment knob

Care of the Microscope

• Use both hands to transport the microscope with one supporting the bottom and the other supporting the neck

• Use lens paper only to wipe the ocular lens, objective lens, condenser

Microorganisms (Microbes)

All organisms that are too small to be seen without a microscope

• Prokaryotes

• Eukaryotes

• Acellular

Prokaryotes

Single-celled organisms characterized by the lack of a nucleus and membrane-bound organelles

• Bacteria (Cyanobacteria)

• Archaea

Eukaryotes

Multi-celled organisms containing a membrane-bound nucleus and organelles like mitochondria

• Algae

• Fungi

• Protozoa

• Helminths

Acellular

Entities lacking a cellular structure, meaning they are not composed of cells, possess no organelles, ribosomes, or plasma membranes

• Viruses

• Viroids

• Prions

Prokarya-Cyanobacteria

Formerly known as blue-green algae, aquatic, photosynthetic, no motility(glide with water currents), reproduce asexually (binary fission), and do not cause infectious disease

• Anabaena

• Oscillatoria

Anabaena

This cyanobacteria formulates a bead-like chain

Oscillatoria

The cyanobacteria formulates long, skinny rectangular strips

Eukarya-Protists-Algae (Green Algae)

This type of microbe is eukaryotic ranging from unicellular to giant multicellular organisms, aquatic, photosynthetic (chloroplasts), motile by flagella, mostly produces asexually, and does not cause infectious disease

• Euglena

• Volvox

• Spirogyra

Flagella

Green algae is motile through the use of this structure

Euglena

This green algae is an elongated oval shape with internal organelles present

Volvox

This green algae has a large, circular shape with internal organelles present

Spirogyra

This green algae is shaped into long rectangles, with a spiral pattern running through it

Protozoa (Ciliates)

This microbe is eukaryotic but is unicellular, lacks a cell wall, thrives in moist environments, heterotrophic, motile by cilia pseudopodia or flagella, mostly reproduces asexually (mitosis), and most do not cause disease

• Paramecium

• Stentor

• Amoebae

• Flagellates

• Apicomplexans

Heterotrophic

An organism that cannot produce its own food, obtaining energy and organic carbon by consuming other organisms, plants, or organic matter.

Paramecium

This protozoa (ciliate) are slipper shaped and travel using cilia

Cilia

Hair-like, microtubule-based organelles on eukaryotic cells that facilitate locomotion, sensory functions, and fluid movement

Flagella

Long, whip-like appendages in microbiology that function as rotary motors, enabling bacterial motility for navigating environments, seeking nutrients, and enhancing virulence

Stentor

This protozoa can either look like venus fly traps or trumpet shaped, are motile using cilis

Amoebae

A blob-like shape, uses pseudopodia for motility

Pseudopodia

Temporary cytoplasmic extensions used for locomotion and feeding (phagocytosis)

Fungi

Eukaryotic ranging from unicellular (yeasts) to filamentous (molds) tp multicellular (mushrooms), heterotrophic, have a cell wall, reproduce asexually (spores) or sexually (zygospore)

Mold Characteristics

• Hyphae

• Mycelium

• Septa

Hyphae

Long, branching, filamentous structures that form the main vegetative growth of fungi

Mycelium

Mass of hyphae

Septa

Thin, dividing walls or membranes that separate cavities, tissues, or cells within organisms, providing structural support, compartmentalization, and regulation of material flow

Asexual Reproduction

Mitotic division of parental cells

Sexual Reproduction

Union of two parental nuclei followed by meiosis

Sporangium

This mode of asexual production is a sac-like structure containing spores

• Rhizopus

• Aspergillus

Phalospores

This mode of asexual reproduction is flask-shaped containing spores

• Penicillium

Zygospore (Sexual)

This mode of sexual reproduction is a bow-like structure with a zygote in the middle

• Rhizopus

Rhizopus Mold (Asexual)

This type of fungi is lollipop shaped and is asexual; reproduce through sporangiospores

Rhizopus Mold (Sexual)

This type of fungi is similarly shaped as round in the center with two tails on each end; reproduce through zygospores

Penicillium Mold (Asexual)

This type of fungi are finger-like shaped; reproduce through phaliospores

Aspergillus Mold (Asexual)

This type of fungi has a stalk followed by a spherical tip that’s mop-like; reproduce from sproangiospores

Yeast (Fungi)

Unicellular, some can form pseudohyphae

Blastospores

asexual fungal spores produced by budding; small daughter cell (bud) grows directly out of a larger mother cell.

Pseudohyphae

Chains of elongated yeast cells that remain attached after budding, forming filaments with distinct constrictions

Candida

Long filaments with budding all around, reproduce through asexual budding

Bacilli

• Round or tapered ends

• Singular or in chains

• Can be motile with flagella

Strepto

Bacteria shape is in a chain

Staphyl

Bacteria shape is in clusters

Cocci

• spherical bacteria shape that can be presented in chains, clusters(diploid, tetrads, many)

• Not motile —> no flagella

Spirals

Curved rods

• Slender = spirochetes

• Thicker = spirilium

• Comma shaped rod = vibrio

• Can be motile with flagella

Turbidity

The cloudiness or haziness of a liquid culture, indicating the presence of bacteria cells

Colony

A visible cluster of microorganisms, often originating from a single cell, growing on solid medium

Aseptic Technique

Set of procedures performed to minimize contamination by microbes/pathogens

• Work area disinfection

• loop and needles

• culture tube flame and inoculation

• petri plate inoculation

Smear Preparation Purpose

• Adhere cells to slide so they do not get washed off by staining steps

• Does not change morphology of cells

• Must be thin enough to allow light to penetrate, allow visualization of individual cells, not allow for trapping of dye

Smear Preparation Steps

1.) Prepare slides (label + draw target circle)

2a.) From liquid —> place one loopful on slide and spread over target circle

2b.) From solid(plate, slant, solid surface) —> place one loop of water on slide and then place small amount of culture in water on slide and spread over target circle

3.) Allow slide to air dry

4.) Pass slide through flame 3-5 times

• kills organisms

• fixes organism to slide

Simple Stain Purpose

• Single, positively charged stains adhere to negatively charged cells

• Ex: methylene blue, fuchsin, crystal violet

• Determine c\shape of bacteria

• Arrangement of bacteria

• Presence of granules

Simple Stain Steps

1.) Use a prepared smear

2.) Add simple stain to sample

3.) Incubate 30 seconds to 2 minutes

4.) Remove excess stain with water

5.) Blot water with bibulous paper

6.) Observe under microscope

Spore Staining Steps

1.) Prepare smear

2.) Cover with paper towel soaked in malachite green dye

3.) Steam over boiling water for 5 minutes

4.) Counterstain with safranin

*Malachite green retained in endospore, vegetative/growing cells stain red/pink with safranin

Acid Fast Bacteria

This type of bacteria contains a high lipid content in their cell wall (waxy) which affects staining abilities

-TB, leprosy

Acid Fast Staining Method

1.) Prepare heat fixed smear

2.) Cover smear with carbolfuchsin for 5 minutes

3.) Wash with water, shake excess water

4.) decolorize with acid-alcohol

5.) Rinse off decolorization after 10 seconds briefly with water

6.) Counterstain with methylene blue

*Acid-fast bacteria will stain red/pink and non-acid bacteria will stain blue

Meningitis

This disease can be found in negative stains

Gram Stain Purpose

The purpose of this stain is to to rapidly classify bacteria into two broad categories based on the structural differences in their cell walls. It provides immediate information on bacterial shape, size, and cell wall composition, allowing clinicians to guide early, targeted antibiotic treatment for infections.

Gram Stain Steps

1.) Prepare heat fixed smear

2.) Apply crystal violet for 1 minute

3.) Wash until stain is removed and shake off excess water

4.) Apply gram’s iodine for 1 minute

5.) Wash off using water

6.) Decolorize for 10 seconds

7.) Wash

8.) Apply safranin for 1 minute

9.) Wash

10.) Blot dry with bibulous paper

11.) Observe

C. diff

A gram stain is used to test for this disease