Name: ________________________
Biology Lab: Mr. Werner
Date: ___________________
Period: __________________
Virtual Bacterial Identification Lab
I. Objectives

To familiarize you with the science and techniques used to identify different types
of bacteria based on their DNA sequence

To work together with a partner to reveal the materials and procedures involved in a
professional laboratory designed to identify bacterial DNA.
II. Process Skills

Comprehending


Interpreting
Analyzing
III. Materials



Laptop
Notebook
HHMI website (http://www.hhmi.org/biointeractive/vlabs/bacterial_id/index.html)
IV. Background
1. Not long ago, DNA sequencing was a time-consuming, tedious process. With readily
available commercial equipment and kits, it is now routine. The techniques used in this lab
are applicable in a wide variety of settings, including scientific research and forensic labs.
Basic Steps




Prepare a sample from a patient and isolate whole bacterial DNA.
Make many copies of the desired piece of DNA.
Sequence the DNA.
Analyze the sequence and identify the bacteria.
2. The segment of DNA used for identifying bacteria is the region that codes for a small
subunit of the ribosomal RNA (16S rRNA). We will refer to this piece as 16S rDNA.
Different bacterial species have unique 16S rDNA sequences. The identification relies on
matching the sequence from your sample against a database of all known 16S rDNA
sequences.
V. Procedure
1. Throughout each exercise, the window will display information explaining what you are
doing. All the interactions, however, will be done inside the graphic window to the left. The
small white box below the graphic will give you specific instructions on what objects to
click on.
2. Below is a list of each of the six parts of the lab. Work through each carefully, reading
the information to the right of the procedure.
Part
Part
Part
Part
Part
Part
1:
2:
3:
4:
5:
6:
Bacterial Sample Preparation
PCR Amplification
PCR Purification
Sequencing Preparation
DNA Sequencing
DNA Sequence Analysis
VI. Analysis and Conclusions-Part A
(1) For each of the terms below, explain the exhibited ROLE or PURPOSE as observed in
the virtual laboratory process. Use the reading and your observations as a guide in
constructing meanings behind each term.
Part 1: Bacterial Sample Preparation:
(a) Gloves: _________________________________________________________
_______________________________________________________________
(b) Inoculating loop: __________________________________________________
_______________________________________________________________
(c) Nutrient culture: __________________________________________________
__________________________________________________________________
(d) Microcentrifuge tube: ______________________________________________
__________________________________________________________________
(e) Orange tray: _____________________________________________________
__________________________________________________________________
(f) Digestive enzymes: ________________________________________________
__________________________________________________________________
(g) Digestive buffer: __________________________________________________
__________________________________________________________________
(h) Micropipette: ____________________________________________________
__________________________________________________________________
(i) Biohazard container: ________________________________________________
__________________________________________________________________
(j) Heated water bath: ________________________________________________
__________________________________________________________________
(k) Centrifuge: ______________________________________________________
__________________________________________________________________
(l) Counterbalance: ___________________________________________________
__________________________________________________________________
(m) Supernatant: ____________________________________________________
__________________________________________________________________
(n) PCR tube: _______________________________________________________
__________________________________________________________________
Part 2: PCR Amplification
(a) PCR master mix solution: ____________________________________________
__________________________________________________________________
(b) Green-capped positive control bottle: ___________________________________
__________________________________________________________________
(c) Deionized water bottle: _____________________________________________
__________________________________________________________________
(d) PCR machine: _____________________________________________________
__________________________________________________________________
Part 3: PCR Purification
(a) Microconcentrator column: __________________________________________
_________________________________________________________________
(b) Yellow-capped buffer bottle: ________________________________________
_________________________________________________________________
(c) PCR product: _____________________________________________________
_________________________________________________________________
(d) Ice bath: _______________________________________________________
_________________________________________________________________
(e) Centrifuge: ______________________________________________________
_________________________________________________________________
(f) New collection tube: _______________________________________________
_________________________________________________________________
(g) Buffer solution: __________________________________________________
_________________________________________________________________
(h) Blue-capped distilled water: _________________________________________
_________________________________________________________________
(i) Green and blue strip tubes: __________________________________________
_________________________________________________________________
Part 4: Sequencing Preparation
(No terms)
Part 5: DNA Sequencing
(a) Automatic Sequencer: ______________________________________________
__________________________________________________________________
Part 6: DNA Sequence Analysis
(a) Genbank: ________________________________________________________
__________________________________________________________________
(b) BLAST: _________________________________________________________
__________________________________________________________________
(c) NCBI site: _______________________________________________________
__________________________________________________________________
VI. Analysis and Conclusions-Part B
(1) What are primers? ___________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
(2) What types of patient samples are used for the purpose of identifying possible
pathogens? ___________________________________________________________
____________________________________________________________________
____________________________________________________________________
(3) What does PCR do, how does it work, and why is it useful? ______________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
(4) How do you separate the desired DNA from all others? ________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
(5) How does an automatic DNA sequencer work? _______________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
(6) How is it possible to use a DNA sequence to identify bacteria? __________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
(7) According to your findings, which species of infectious bacteria did your patient suffer
from? _______________________________________________________________
____________________________________________________________________
____________________________________________________________________
(8) Finally, provide a brief synopsis of the major features that were involved behind each
of the following parts of the lab. What were the overall objectives or key points that
were recognized during each stage?
Part 1: Bacterial Sample Preparation
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Part 2: PCR Amplification
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Part 3: PCR Purification
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Part 4: Sequencing Preparation
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Part 5: DNA Sequencing
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Part 6: DNA Sequence Analysis
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________